Methods for monitoring multiple gene expression

ABSTRACT

The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing  Trichoderma reesei  ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 10/950,009, filed Sep. 24, 2004, which claims the benefit of U.S. Provisional Application No. 60/506,140, filed Sep. 25, 2003, which applications are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under NREL Subcontract No. ZCO-30017-02, Prime Contract DE-AC36-98GO10337 awarded by the Department of Energy. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for monitoring expression of a plurality of genes in filamentous fungal cells. The present invention also relates to substrates and computer readable media for monitoring expression of a plurality of genes in filamentous fungal cells.

2. Description of the Related Art

Microarray technology is increasingly becoming the method of choice for the quantitative and simultaneous analysis of the expression levels of many thousands of genes. Microarray analyses typically follow the steps of gene selection, microarray synthesis, sample preparation, array hybridization, detection, and data analysis (Watson et al., 1998, Current Opinion in Biotechnology 9: 609-614).

PCR-amplified coding sequences of genomic DNA are particularly useful in microarrays for obtaining global expression profiles where the genome of the organism has been fully sequenced. Chu et at, 1998, Science 282: 699-705, disclose the use of microarrays containing PCR-amplified genomic coding sequences for determining the temporal expression of Saccharomyces cerevisiae genes during sporulation.

For other organisms whose genomes have not been sequenced, global expression profiles may be obtained with arraying (1) random genomic DNA segments or clones (e.g., from a genomic DNA library); (2) random cDNA clones (e.g., from one or more cDNA libraries) that are uncharacterized at the DNA sequence level; (3) EST clones that have been sequenced and partially characterized with respect to putative identification and function; or (4) cDNA clones that are enriched for differentially expressed sequences (Diatchenko et al., 1996, Proc. Natl. Acad. Sci. USA 93: 6025-6030).

However, there are disadvantages with using random genomic or cDNA clones from organisms whose genomes have not been fully sequenced. These disadvantages include (1) more than one gene may be represented on a single clone; (2) no gene(s) may be encoded on a single clone; (3) extensive characterization and DNA sequencing is required to follow-up array spots that appear interesting; and (4) duplicity, multiplicity, and redundancy add to the follow-up work.

Expressed sequenced tags (ESTs) are partial cDNA sequences of expressed genes. Simply stated, an EST is a segment of a sequence from a cDNA clone that corresponds to the mRNA of a specific gene. The use of sequenced ESTs in microarrays compared to genomic clones or random cDNA clones provides several advantages especially for organisms whose genomes have not been sequenced. First, redundancy is eliminated because one spot on an array equals one gene or open reading frame. Second, since sequence information is available, redundancy and follow-up characterization is minimized. Third, EST microarrays can be organized based on function of the gene products to facilitate analysis of the results (e.g., ESTs encoding enzymes from the same metabolic pathway can be arranged or grouped accordingly).

Ruan et al., 1998, The Plant Journal 15: 821-833, disclose the use of microarrays containing Arabidopsis thaliana EST sequences for determining the temporal expression of Arabidopsis thaliana genes in root, leaf, and two stages of floral development.

Iyer et al., 1999, Science 283; 83-87, disclose the use of microarrays containing human EST sequences for determining the temporal expression of human fibroblast cells in response to serum.

Diatchenko et al., 1996, supra, disclose a method called suppression subtractive hybridization (SSH) for generating differentially regulated or tissue-specific cDNA probes and libraries. Yang et al., 1999, Nucleic Acids Research 27: 1517-1523, describe combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Porkka and Visakorpi, 2001, Journal of Pathology 193: 73-79, disclose detection of differentially expressed genes in prostrate cancer by combining SSH and cDNA library arrays.

Hayward et al., 2000, Molecular Microbiology 35: 6-14, disclose shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria.

WO 2000/56762 discloses methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing filamentous fungal expressed sequenced tags.

Filamentous fungi are increasingly being used as host microorganisms for the industrial production of enzymes and other proteins whether endogenous or heterogenous to the microorganisms. There is a need in the art to provide methods for monitoring the global expression of genes from filamentous fungal cells to improve the production potential of these microorganisms and to identify new genes whose products have industrial applicability.

It is an object of the present invention to provide alternative methods for monitoring expression of a plurality of genes in filamentous fungal cells.

SUMMARY OF THE INVENTION

The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells, comprising:

(a) adding a mixture of detection reporter-labeled nucleic acids isolated from the filamentous fungal cells to a substrate containing an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, and nucleic acid sequences having at least 90% homology to SEQ ID NOs. 1-1188, under conditions where the nucleic acids hybridize to complementary sequences of the ESTs or SSH clones, or a combination thereof, in the array, wherein the nucleic acids from the first filamentous fungal cell and the one or more second filamentous fungal cells are labeled with a first detection reporter and one or more different second detection reporters, respectively; and

(b) examining the array under conditions wherein the relative expression of the genes in the filamentous fungal cells is determined by the observed detection signal of each spot in the array in which (i) the Trichoderma reesei ESTs or SSH clones, or a combination thereof, in the array that hybridize to the nucleic acids obtained from either the first or the one or more second filamentous fungal cells produce a distinct first detection signal or one or more second detection signals, respectively, and (ii) the Trichoderma reesei ESTs or SSH clones, or a combination thereof, in the array that hybridize to the nucleic acids obtained from both the first and one or more second filamentous fungal cells produce a distinct combined detection signal.

The present invention also relates to computer readable media and substrates containing an array of such Trichoderma reesei ESTs or SSH clones, or a combination thereof, for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells.

DESCRIPTION OF FIGURE

FIG. 1 show the SSH method for generation of subtractive and normalized cDNA libraries.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells. The methods comprise (a) adding a mixture of detection reporter-labeled nucleic acids isolated from the two or more filamentous fungal cells with different detection reporters for each cell's nucleic acids to a substrate containing an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, under conditions where the nucleic acids hybridize to complementary sequences of the ESTs or SSH clones, or a combination thereof, in the array; and (b) examining the array under conditions wherein the relative expression of the genes in the two or more cells is determined by the observed detection signal of each spot in the array.

The methods of the present invention may be used to monitor global expression of a plurality of genes from a filamentous fungal cell, discover new genes, identify possible functions of unknown open reading frames, and monitor gene copy number variation and stability. For example, the global view of changes in expression of genes may be used to provide a picture of the way in which filamentous fungal cells adapt to changes in culture conditions, environmental stress, or other physiological provocation. Other possibilities for monitoring global expression include spore morphogenesis, recombination, metabolic or catabolic pathway engineering. In a preferred embodiment, the methods of the present invention are used to identify microbial genes induced when the microorganism is grown on cellulose or corn stover. In more preferred embodiment, the microorganism is a Trichoderma strain. In a most preferred embodiment, the microorganism is a Trichoderma reesei strain.

The methods of the present invention are particularly advantageous because one spot on an array equals one gene or open reading frame, extensive follow-up characterization is unnecessary since sequence information is available, and EST and/or SSH microarrays can be organized based on function of the gene products.

Expressed Sequenced Tags and Suppression Subtractive Hybridization (SSH) Clones

The term “array features” is defined herein as array elements of ESTs or SSH clones, or a combination thereof.

The term “expressed sequenced tag” or “EST” is defined herein as a segment of a sequence from a cDNA clone of an expressed Trichoderma reesei gene. The term “EST” will be understood to also include two or more ESTs assembled into a contig. In the methods of the present invention, the Trichoderma reesei ESTs described herein preferably represent a plurality of genes or homologues thereof present in the two or more filamentous fungal cells to be evaluated.

ESTs are generally generated as follows: Total polyadenylated mRNA is isolated from a filamentous fungal cell and reverse transcribed into total cDNA. The total cDNA is digested with a restriction endonuclease, size-selected by agarose gel electrophoresis, isolated, and ligated into a vector, e.g., pZErO-2.1. The ligation mixture is transformed into competent E. coli cells and transformants are selected under selective pressure, e.g., kanamycin selection. The cDNA clones isolated from the selected transformants are amplified, isolated, and partially sequenced. The partial sequences are then compared to sequences in various publicly available databases for identification.

Any method known in the art may be used for generating ESTs (see, for example, Adams et al., 1991, Science 252: 1651-1656; Fields, 1996, Tibtech 14: 286-289; Weinstock et al., 1994, Current Opinion in Biotechnology 5: 599-603; Matsubara and Okubo, 1993, Current Opinions in Biotechnology 4: 672-677; Nelson et al., 1997, Fungal Genet. Biol. 21: 348-363; Zhu at al., Genetics 157: 1057-1065).

In a preferred embodiment, the ESTs are SEQ ID NOs: 1-24.

The term “SSH clones” is defined herein as selectively amplified target cDNA fragments which are differentially expressed. SSH is used to selectively amplify these target cDNA fragments and simultaneously suppress nontarget DNA amplification.

Any method known in the art may be used for generating SSH clones (see, for example, Diatchenko et al., 1996, supra; Yang et al, 1999, supra; Porkka and Visakorpi, 2001, supra).

In a preferred embodiment, the SSH clones are SEQ ID NOs: 25-65.

In the methods of the present invention, the Trichoderma reesei array features are preferably at least about 50 bp in length, more preferably at least about 100 bp in length, even more preferably at least about 150 bp in length, and most preferably at least about 200 bp in length. Furthermore, the array features are preferably directional ESTs or SSH clones, or a combination thereof. However, nondirectional ESTs or SSH clones, or a combination thereof, may also be used. A “directional EST” is defined as a cDNA cloned in the same orientation relative to the vector cloning sites, e.g., 5′→3′ or 3′→5′.

In a preferred embodiment, the array features are obtained from Trichoderma reesei. In a more preferred embodiment, the array features are obtained from Trichoderma reesei strain RutC30 (Montenecourt and Eveleigh, 1979, Adv. Chem. Ser. 181: 289-301). In a most preferred embodiment, the Trichoderma reesei array features are selected from the group consisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, or nucleic acid sequences having at least 95%, preferably at least 99% and most preferably at least 99.9% homology to a sequence of SEQ ID NOs. 1-1188.

In another preferred embodiment, the array features obtained from Trichoderma reesei are ESTs. In another preferred embodiment, the array features obtained from Trichoderma reesei are SSH clones. In another preferred embodiment, the array features obtained from Trichoderma reesei are a combination of two or more of ESTs and SSH clones.

For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, and windows=20.

Microarrays

The term “an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof” is defined herein as a linear or two-dimensional array of preferably discrete array features, each having a finite area, formed on the surface of a solid support.

The term “microarray” is defined herein as an array of features (i.e., ESTs or SSH clones, or a combination thereof) having a density of discrete array elements of at least about 100/cm², and preferably at least about 1000/cm². The printed elements in a microarray have typical dimensions, e.g., diameters, in the range of between about 10 to about 250 μm, preferably in the range of between about 10 to about 200 μm, more preferably in the range of between about 20 to about 150 μm, even more preferably in the range of between about 20 to about 100 μm, most preferably in the range of between about 20 to about 75 μm, and even most preferably in the range of between about 25 to about 50 μm, and are separated from other printed elements in the microarray by about the same distance.

Methods and instruments for forming microarrays on the surface of a solid support are well known in the art. See, for example, U.S. Pat. No. 5,807,522; U.S. Pat. No. 5,700,637; and U.S. Pat. No. 5,770,151. The instrument may be an automated device such as described in U.S. Pat. No. 5,807,522.

The term “a substrate containing an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof,” is defined herein as a solid support having deposited on the surface of the support one or more of a plurality of array features, for use in detecting binding of labeled cDNAs to the array features.

The substrate may, in one aspect, be a glass support (e.g., glass slide) having a hydrophilic or hydrophobic coating on the surface of the support, and an array of distinct array features electrostatically bound non-covalently to the coating, where each distinct array features is disposed at a separate, defined position.

Each microarray in the substrate preferably contains at least 10³ distinct array features in a surface area of less than about 1 cm². Each distinct array feature (i) is disposed at a separate, defined position in the array, (ii) has a length of at least 50 bp, and (iii) is present in a defined amount between about 0.1 femtomoles and 100 nanomoles or higher if necessary.

For a hydrophilic coating, the glass slide is coated by placing a film of a polycationic polymer with a uniform thickness on the surface of the slide and drying the film to form a dried coating. The amount of polycationic polymer added should be sufficient to form at least a monolayer of polymers on the glass surface. The polymer film is bound to the surface via electrostatic binding between negative silyl-OH groups on the surface and charged cationic groups in the polymers. Such polycationic polymers include, but are not limited to, polylysine and polyarginine.

Another coating strategy employs reactive aldehydes to couple DNA to the slides (Schena et al., 1996, Proceedings of the National Academy of Science USA 93: 10614-10619; Heller at al., 1997, Proceedings of the National Academy of Science USA 94: 2150-2155).

Alternatively, the surface may have a relatively hydrophobic character, one that causes aqueous medium deposited on the surface to bead. A variety of known hydrophobic polymers, such as polystyrene, polypropylene, or polyethylene, have desirable hydrophobic properties, as do glass and a variety of lubricant or other hydrophobic films that may be applied to the support surface. A support surface is “hydrophobic” if an aqueous droplet applied to the surface does not spread out substantially beyond the area size of the applied droplet, wherein the surface acts to prevent spreading of the droplet applied to the surface by hydrophobic interaction with the droplet.

In another aspect, the substrate may be a multi-cell substrate where each cell contains a microarray of array features, and preferably an identical microarray, formed on a porous surface. For example, a 96-cell array may typically have array dimensions between about 12 and 244 mm in width and 8 and 400 mm in length, with the cells in the array having width and length dimension of l/12 and l/8 the array width and length dimensions, respectively, i.e., between about 1 and 20 in width and 1 and 50 mm in length.

The solid support may include a water-impermeable backing such as a glass slide or rigid polymer sheet, or other non-porous material. Formed on the surface of the backing is a water-permeable film which is made of porous material. Such porous materials include, but are not limited to, nitrocellulose membrane nylon, polypropylene, and PVDF polymer. The thickness of the film is preferably between about 10 and 1000 μm. The film may be applied to the backing by spraying or coating, or by applying a preformed membrane to the backing.

The film surface may be partitioned into a desirable array of cells by water-impermeable grid lines typically at a distance of about 100 to 2000 μm above the film surface. The grid lines can be formed on the surface of the film by laying down an uncured flowable resin or elastomer solution in an array grid, allowing the material to infiltrate the porous film down to the backing, and then curing the grid lines to form the cell-array substrate.

The barrier material of the grid lines may be a flowable silicone, wax-based material, thermoset material (e.g., epoxy), or any other useful material. The grid lines may be applied to the solid support using a narrow syringe, printing techniques, heat-seal stamping, or any other useful method known in the art.

Each well preferably contains a microarray of distinct array features. “Distinct array features” as applied to the ESTs or SSH clones, or a combination thereof, forming a microarray is defined herein as an array feature which is distinct from other array features on the basis of a different nucleic acid sequence, and/or different concentrations of the same or distinct array features, and/or different mixtures of distinct array features or different-concentrations of array features. Thus an array of “distinct array features” may be an array containing, as its components, (i) distinct array features, which may have a defined amount in each component, (ii) different, graded concentrations of given-sequence array features, and/or (iii) different-composition mixtures of two or more distinct array features.

However, any type of substrate known in the art may be used in the methods of the present invention.

The delivery of a known amount of a selected EST or SSH clone to a specific position on the support surface is preferably performed with a dispensing device equipped with one or more tips for insuring reproducible deposition and location of the array features and for preparing multiple arrays. Any dispensing device known in the art may be used in the methods of the present invention. See, for example, U.S. Pat. No. 5,807,522. The dispensing device preferably contains a plurality of tips.

For liquid-dispensing on a hydrophilic surface, the liquid will have less of a tendency to bead, and the dispensed volume will be more sensitive to the total dwell time of the dispenser tip in the immediate vicinity of the support surface.

For liquid-dispensing on a hydrophobic surface, flow of fluid from the tip onto the support surface will continue from the dispenser onto the support surface until it forms a liquid bead. At a given bead size, i.e., volume, the tendency of liquid to flow onto the surface will be balanced by the hydrophobic surface interaction of the bead with the support surface, which acts to limit the total bead area on the surface, and by the surface tension of the droplet, which tends toward a given bead curvature. At this point, a given bead volume will have formed, and continued contact of the dispenser tip with the bead, as the dispenser tip is being withdrawn, will have little or no effect on bead volume.

The desired deposition volume, i.e., bead volume, formed is preferably in the range 2 pl (picoliters) to 2 nl (nanoliters), although volumes as high as 100 nl or more may be dispensed. It will be appreciated that the selected dispensed volume will depend on (i) the “footprint” of the dispenser tip(s), i.e., the size of the area spanned by the tip(s), (ii) the hydrophobicity of the support surface, and (iii) the time of contact with and rate of withdrawal of the tip(s) from the support surface. In addition, bead size may be reduced by increasing the viscosity of the medium, effectively reducing the flow time of liquid from the dispensing device onto the support surface. The drop size may be further constrained by depositing the drop in a hydrophilic region surrounded by a hydrophobic grid pattern on the support surface.

At a given tip size, bead volume can be reduced in a controlled fashion by increasing surface hydrophobicity, reducing time of contact of the tip with the surface, increasing rate of movement of the tip away from the surface, and/or increasing the viscosity of the medium. Once these parameters are fixed, a selected deposition volume in the desired pl to nl range can be achieved in a repeatable fashion.

After depositing a liquid droplet of an array feature sample at one selected location on a support, the tip may be moved to a corresponding position on a second support, the sample is deposited at that position, and this process is repeated until the sample has been deposited at a selected position on a plurality of supports.

This deposition process may then be repeated with another EST or SSH clone sample at another microarray position on each of the supports.

The diameter of each array feature region is preferably between about 20-200 μm. The spacing between each region and its closest (non-diagonal) neighbor, measured from center-to-center, is preferably in the range of about 20-400 μm. Thus, for example, an array having a center-to-center spacing of about 250 μm contains about 40 regions/cm² or 1,600 regions/cm². After formation of the array, the support is treated to evaporate the liquid of the droplet forming each region, to leave a desired array of dried, relatively flat array feature regions. This drying may be done by heating or under vacuum.

Filamentous Fungal Cells

In the methods of the present invention, the two or more filamentous fungal cells may be any filamentous fungal cell where one of the cells is used as a reference for identifying differences in expression of the same or similar complement of genes in the other cell. In one aspect, the two or more cells are the same cell. For example, they may be compared under different growth conditions, e.g., carbon source, oxygen limitation, nutrition, and/or physiology. In another aspect, one or more cells are mutants of the reference cell. For example, the mutant(s) may have a different phenotype. In a further aspect, the two or more cells are of different species (e.g., Trichoderma reesei and Trichoderma viride). In another further aspect, the two or more cells are of different genera. In an even further aspect, one or more cells are transformants of the reference cell, wherein the one or more transformants exhibit a different property. For example, the transformants may have a different or improved phenotype relative to the reference cell and/or one of the other transformants. The term “phenotype” is defined herein as an observable or outward characteristic of a cell determined by its genotype and modulated by its environment. Such different or improved phenotypes may include, but are not limited to, improved secretion or production of a protein or compound, reduced or no secretion or production of a protein or compound, improved or reduced expression of a gene, desirable morphology, an altered growth rate under desired conditions, relief of over-expression mediated growth inhibition, or tolerance to low oxygen conditions, improved filterability or flocculation properties, or altered protein glycosylation.

In a preferred embodiment, the differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells is a result of growth of the first filamentous fungal cell on glucose and growth of the one or more second filamentous fungal cells on cellulose, hemicellulose, and/or corn stover to identify genes that are induced by growth on cellulose, hemicellulose, or corn stover. The corn stover is preferably pre-treated and washed corn stover as described herein.

The filamentous fungal cells may be any filamentous fungal cells, but preferably Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cells, and more preferably Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsatus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichiodes, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

In a preferred embodiment, the filamentous fungal cells are Trichoderma cells. In a more preferred embodiment, the Trichoderma cells are Trichoderma reesei cells. In a most preferred embodiment, the Trichoderma cells are Trichoderma reesei strain RutC30 (Montenecourt and Eveleigh, 1979, supra).

In the methods of the present invention, the cells are cultivated in a nutrient medium suitable for growth using methods well known in the art for isolation of the nucleic acids to be used as probes. For example, the cells may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).

Nucleic Acid Probes

The nucleic acid probes from the two or more filamentous fungal cells may be any nucleic acid including genomic DNA, cDNA, and RNA, and may be isolated using standard methods known in the art. For example, cDNA probes may be obtained from the total polyadenylated mRNA isolated from the cells using standard methods and reverse transcribed into total cDNA.

The populations of isolated nucleic acid probes may be labeled with detection reporters such as colorimetric, radioactive, fluorescent reporters, or other reporters using methods known in the art (Chen et al., 1998, Genomics 51: 313-324; DeRisi et al., 1997, Science 278: 680-686; U.S. Pat. No. 5,770,367).

In a preferred embodiment, the probes are labeled with fluorescent reporters. For example, cDNA probes may be labeled during reverse transcription from the respective mRNA pools by incorporation of fluorophores as dye-labeled nucleotides (DeRisi et al., 1997, supra), e.g., Cy5-labeled deoxyuridine triphosphate, or the isolated cDNAs may be directly labeled with different fluorescent functional groups. Fluorescent-labeled nucleotides include, but are not limited to, fluorescein conjugated nucleotide analogs (green fluorescence) and lissamine nucleotide analogs (red fluorescence). Fluorescent functional groups include, but are not limited to, Cy3 (a green fluorescent dye) and Cy5 (red fluorescent dye).

Array Hybridization

The labeled nucleic acids from the two or more filamentous fungal cells are then added to a substrate containing an array of Trichoderma reesei array features under conditions where the nucleic acid pools from the two or more filamentous fungal cells hybridize to complementary sequences of the array features in the array. For purposes of the present invention, hybridization indicates that the labeled nucleic acids from the two or more cells hybridize to the array features under very low to very high stringency conditions.

A small volume of the labeled nucleic acids mixture is loaded onto the substrate. The solution will spread to cover the entire microarray. In the case of a multi-cell substrate, one or more solutions are loaded into each cell which stop at the barrier elements.

The labeled probes are denatured and applied to a microarray slide under a cover glass, placed in a humidified chamber, and incubated overnight (15-16 hours) in a water bath at 63° C. Before scanning, the arrays are washed consecutively in 1×SSC with 0.03% SDS, 0.2×SSC, and 0.05×SSC and centrifuged for 2 minutes at 500 rpm top remove excess liquid. For further details, see Berka et al., 2003, Proc. Natl. Acad. Sci. USA 100: 5682-5687.

For nucleic acid probes of at least about 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For nucleic acid probes of at least about 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SOS preferably at least at 45° C. (very low stringency), more preferably at least at 50° C. (low stringency), more preferably at least at 55° C. (medium stringency), more preferably at least at 60° C. (medium-high stringency), even more preferably at least at 65° C. (high stringency), and most preferably at least at 70° C. (very high stringency).

For shorter nucleic acid probes which are about 50 nucleotides to about 100 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at 5° C. to 10° C. below the calculated T_(m) using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.

For shorter nucleic acid probes which are about 50 nucleotides to about 100 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated T_(m).

The choice of hybridization conditions will depend on the degree of homology between the Trichoderma reesei array features and the nucleic acids obtained from the two or more filamentous fungal cells. For example, where the cells are the same cell from which the array features were obtained, high stringency conditions may be most suitable. Where the cells are from a genus or species different from which the Trichoderma reesei array features were obtained, low or medium stringency conditions may be more suitable.

In a preferred embodiment, the hybridization is conducted under low stringency conditions. In a more preferred embodiment, the hybridization is conducted under medium stringency conditions. In a most preferred embodiment, the hybridization is conducted under high stringency conditions.

The entire solid support is then reacted with detection reagents, if needed, and analyzed using standard calorimetric, radioactive, or fluorescent detection means. All processing and detection steps are performed simultaneously to all of the microarrays on the solid support ensuring uniform assay conditions for all of the microarrays on the solid support.

Detection

The most common detection method is laser-induced fluorescence detection using confocal optics (Cheung et al, 1998, Nat. Genet. 18: 225-230). The array is examined under fluorescence excitation conditions such that (i) the Trichoderma reesei array features in the array that hybridize to the nucleic acid probes obtained from one of the first cell and one or more second cells produces a distinct first fluorescence emission color or one or second fluorescence emission colors, respectively, and (ii) the Trichoderma reesei ESTs or SSH clones, or a combination thereof, in the array that hybridize to substantially equal numbers of nucleic acid probes obtained from the first cell and one of the one or more second cells produce a distinct combined fluorescence emission color, respectively; wherein the relative expression of the genes in the two or more cells can be determined by the observed fluorescence emission color of each spot in the array.

The fluorescence excitation conditions are based on the selection of the fluorescence reporters. For example, Cy3 and Cy5 reporters are detected with solid state lasers operating at 532 nm and 650 nm, respectively.

However, other methods of detection well known in the art may be used such as standard photometric, calorimetric, or radioactive detection means, as described earlier.

In a preferred embodiment, the methods comprise (a) adding a mixture of fluorescence-labeled nucleic acids isolated from the filamentous fungal cells to a substrate containing an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, and nucleic acid sequences having at least 90% homology to SEQ ID NOs. 1-1188, under conditions where the nucleic acids hybridize to complementary sequences of the ESTs or SSH clones; or a combination thereof; in the array, wherein the nucleic acids from the first filamentous fungal cell and the one or more second filamentous fungal cells are labeled with a first fluorescent reporter and one or more different second fluorescent reporters, respectively; and (b) examining the array by fluorescence under fluorescence excitation conditions wherein the relative expression of the genes in the filamentous fungal cells is determined by the observed fluorescence emission color of each spot in the array in which (i) the Trichoderma reesei ESTs or SSH clones, or a combination thereof, in the array that hybridize to the nucleic acids obtained from either the first or the one or more second filamentous fungal cells produce a distinct first fluorescence emission color or one or more second fluorescence emission colors, respectively, and (ii) the Trichoderma reesei ESTs or SSH clones; or a combination thereof; in the array that hybridize to the nucleic acids obtained from both the first and one or more second filamentous fungal cells produce a distinct combined fluorescence emission color.

Data Analysis

The data obtained from the scanned image may then be analyzed using any of the commercially available image analysis software. The software preferably identifies array elements, subtracts backgrounds, deconvolutes multi-color images, flags or removes artifacts, verifies that controls have performed properly, and normalizes the signals (Chen et al, 1997, Journal of Biomedical Optics 2: 364-374).

Several computational methods have been described for the analysis and interpretation of microarray-based expression profiles including cluster analysis (Eisen et al, 1998, Proc. Nat. Acad. Sol USA 95: 14863-14868), parametric ordering of genes (Spellman et al., 1998, Mol. Biol. Cell 9: 3273-3297), and supervised clustering methods based on representative hand-picked or computer-generated expression profiles (Chu et al, 1998. Science 282: 699-705). Preferred methods for evaluating the results of the microarrays employ statistical analysis to determine the significance of the differences in expression levels. In the methods of the present invention, the difference in the detected expression level is at least about 10% or greater, preferably at least about 20% or greater, more preferably at least about 50% or greater, even more preferably at least about 75% or greater; and most preferably at least about 100% or greater.

One such preferred system is the Significance Analysis of Microarrays (SAM) (Tusher et al, 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121). Statistical analysis allows the determination of significantly altered expression of levels of about 50% or even less. The PAM (or predictive analysis for microarrays) represents another approach for analyzing the results of the microarrays (Tibshirani et al, 2002, Proc. Natl. Acad. Sci. USA 99: 6567-6572).

Cluster algorithms may also be used to analyze microarray expression data. From the analysis of the expression profiles it is possible to identify co-regulated genes that perform common metabolic or biosynthetic functions. Hierarchical clustering has been employed in the analysis of microarray expression data in order to place genes into dusters based on sharing similar patterns of expression (Eisen et al., 1998, supra). This method yields a graphical display that resembles a kind of phylogenetic tree where the relatedness of the expression behavior of each gene to every other gene is depicted by branch lengths. The programs Cluster and TreeView, both written by Michael Eisen (Eisen et al., 1998 Proc. Nat. Acad. Sci. USA 95: 14863-14868) are freely available. Genespring is a commercial program available for such analysis (Silicon Genetics, Redwood City, Calif.).

Self-organizing maps (SOMs), a non-hierarchical method, have also been used to analyze microarray expression data (Tamayo et al, 1999, Proc Natl. Acad. Sci. USA 96: 2907-2912). This method involves selecting a geometry of nodes, where the number of nodes defines the number of clusters. Then, the number of genes analyzed and the number of experimental conditions that were used to provide the expression values of these genes are subjected to an iterative process (20,000-50,000 iterations) that maps the nodes and data points into multidimensional gene expression space. After the identification of significantly regulated genes, the expression level of each gene is normalized across experiments. As a result, the expression profile of the genome is highlighted in a manner that is relatively independent of each gene's expression magnitude. Software for the “GENECLUSTER” SOM program for microarray expression analysis can be obtained from the Whitehead/MIT Center for Genome Research. SOMs can also be constructed using the GeneSpring software package.

Computer Readable Media

The Trichoderma reesei array features described herein may be “provided” in a variety of mediums to facilitate their use. The term “provided” refers to a manufacture comprising an array of Trichoderma array features. Such manufactures provide a large portion of the genomes of Trichoderma reesei and parts thereof (e.g., an open reading frame (ORF)) in a form which allows one skilled in the art to examine the manufacture using means not directly applicable to examining the genome or a subset thereof as it exists in nature or in purified form.

Thus, the present invention also relates to such a manufacture in the form of a computer readable medium comprising an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof. In a preferred embodiment, the computer readable medium comprises an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, or nucleic acid sequences having at least 95%, preferably at least 99% and most preferably at least 99.9% homology to a sequence of SEQ ID NOs. 1-1188. In another preferred embodiment, the computer readable medium comprises an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188.

In one application of this embodiment, the Trichoderma reesei array features can be recorded on computer readable media. The term “computer readable media” is defined herein as any medium which can be read and accessed directly by a computer. Such computer readable media include, but are not limited to, magnetic storage media, e.g., floppy discs, hard disc storage medium, and magnetic tape; optical storage media, e.g., CD-ROM, DVD; electrical storage media, e.g., RAM and ROM; and hybrids of these categories, e.g., magnetic/optical storage media. One skilled in the art can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising computer readable medium having recorded thereon nucleotide sequences of the Trichoderma reesei array features of the present invention. Likewise, it will be clear to those of skill how additional computer readable media that may be developed also can be used to create analogous manufactures having recorded thereon nucleotide sequences of the Trichoderma reesei array features.

As used herein, “recorded” refers to a process for storing information on computer readable medium. One skilled in the art can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.

A variety of data storage structures are available for creating a computer readable medium having recorded thereon the Trichoderma reesei array features of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data-processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

Various computer software are publicly available that allow a skilled artisan to access sequence information provided in a computer readable medium. Thus, by providing in computer readable form an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, and nucleic acid sequences having at least 90%, preferably at least 95%, more preferably at least 99%, and most preferably at least 99.9% homology to SEQ ID NOs. 1-1188 enables one skilled in the art to routinely access the provided sequence information for a wide variety of purposes.

Software utilizing the BLAST (Attschul et al., 1990, Journal of Molecular Biology 215: 403-410) and BLAZE (Brutlag et al., 1993, Comp. Chem. 17: 203-207) search algorithms may be used to identify open reading frames (ORFs) within a genome of interest, which contain homology to ORFs or proteins from Trichoderma reesei and from other organisms. Among the ORFs discussed herein are protein encoding fragments of the Trichoderma reesei genome useful in producing commercially important proteins, such as enzymes, and in the production of commercially useful metabolites.

The present invention further provides systems, particularly computer-based systems, which contain the sequence information described herein. Such systems are designed to identify, among other things, genes and gene products—many of which could be products themselves or used to genetically modify an industrial expression host through increased or decreased expression of a specific gene sequence(s).

The term “a computer-based system” is defined herein as a hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. One skilled in the art can readily appreciate that any currently available computer-based system is suitable for use in the present invention.

As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein nucleic acid sequences of the Trichoderma reesei array features and the necessary hardware means and software means for supporting and implementing a search means.

The term “data storage means” is defined herein as memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.

The term “search means” is defined herein as one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the present sequences which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, MacPattern (Fuchs, 1991, Comput. Appl. Biosci. 7: 105-106), BLASTN and BLASTX (NCBI). One skilled in the art can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.

The term “target sequence” is defined herein as any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. One skilled in the art can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

The term “a target structural motif” or “target motif” is defined herein as any rationally selected sequence or combination of sequences chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences, substrate and cofactor binding domains, transmembrane domains, and sites for post-translational modifications. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences), repeats, palindromes, dyad symmetries, intron-exon boundaries, transcription and translation start and stop sites, and polyadenylation signals.

A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. A preferred format for an output means ranks fragments of the nucleic acid sequences possessing varying degrees of homology to the target sequence or target motif. Such presentation provides one skilled in the art with a ranking of sequences which contain various amounts of the target sequence or target motif and identifies the degree of homology contained in the identified fragment.

A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify sequence fragments of a genome. For example, implementing software which utilize the BLAST and BLAZE algorithms, described in Altschul et al., 1990, Journal of Molecular Biology 215: 403-410, may be used to identify open reading frames within the Trichoderma reesei genome or the genomes of other organisms. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer-based systems of the present invention. Of course, suitable proprietary systems that may be known to those of skill also may be employed in this regard.

Substrates

The present invention also relates to substrates as described herein comprising an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof. In a preferred embodiment, the substrate comprises an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, or nucleic acid sequences having at least 95%, preferably at least 99% and most preferably at least 99.9% homology to a sequence of SEQ ID NOs. 1-1188. In a more preferred embodiment, the substrate comprises an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188.

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Example 1 Fermentation and Mycelial Tissue

Trichoderma reesei strain RutC30 (Montenecourt and Eveleigh, 1979, Adv. Chem. Ser. 181: 289-301) was cultivated in a pilot scale fermentation tank in growth medium containing a complex carbon source. The carbon sources included glucose, cellulose, or pre-treated and washed corn stover. Fungal mycelium was collected from a one-liter sample, and immediately frozen in liquid N₂ and stored at −80° C.

Pretreated corn stover (PCS) was obtained from U.S. Department of Energy National Renewable Energy Laboratory (NREL). The water-insoluble solids in PCS include: 56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin. Pretreatment conditions were: corn stover, 1.4% (wt/vol) sulfuric acid, 165° C., 107 psi, for 8 minutes. Prior to assay, PCS was washed with a large volume of distilled deionized water on a glass filter. PCS was then milled using a coffee grinder to reduce particle size, then washed further with water on a 22 Millipore filter (6P Express Membrane, Stericup, Millipore, Billerica, Mass.). The washed PCS was resuspended in deionized water to make a 20 mg/ml suspension, and stored at 4° C.

Example 2 Trichoderma reesei Directional cDNA Library Construction

Total RNA was prepared from the Trichoderma reesei mycelial samples described in Example 1 by extraction with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion (Chirgwin et al, 1979, Biochemistry 18: 5294-5299) using the following modifications. The frozen mycelia were ground in liquid N₂ to a fine powder with a mortar and a pestle, followed by grinding in a precooled coffee mill, and immediately suspended in 5 volumes of RNA extraction buffer (4 M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, 25 mM sodium citrate pH 7.0, 0.1 M β-mercaptoethanol). The mixture was stirred for 30 minutes at room temperature and centrifuged (20 minutes at 12,000×g) to pellet the cell debris. The supernatant was collected, carefully layered onto a 5.7 M CsCl cushion (5.7 M CsCl, 10 mM EDTA, pH 7.5, 0.1% DEPC; autoclaved prior to use) using 26.5 ml supernatant per 12.0 ml of CsCl cushion, and centrifuged to obtain the total RNA (Beckman SW 28 rotor, 25,000 rpm, room temperature, 24 hours). After centrifugation the supernatant was carefully removed and the bottom of the tube containing the RNA pellet was cut off and rinsed with 70% ethanol. The total RNA pellet was transferred to an Eppendorf tube, suspended in 500 μl of TE (10 mM Tris-0.1 mM EDTA), pH 7.6 (if difficult, heated occasionally for 5 minutes at 65° C.), phenol extracted, and precipitated with ethanol for 12 hours at −20° C. (2.5 volumes of ethanol, 0.1 volume of 3M sodium acetate pH 5.2). The RNA was collected by centrifugation (30 minutes at 12,000×g), washed in 70% ethanol, and resuspended in a minimum volume of DEPC-treated water. The total RNA concentration was determined by measuring the absorbance at 260 nm.

Poly(A)⁺ RNA was isolated by oligo(dT)-cellulose affinity chromatography (Aviv & Leder, 1972, Proceedings of the National Academy of Sciences USA 69: 1408-1412). A total of 0.2 g of oligo(dT) cellulose (Boehringer Mannheim, Indianapolis, Ind.) was pre-swollen in 10 ml of 1× of column loading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS), loaded onto a DEPC-treated, plugged plastic column (Poly Prep Chromatography Column, BioRad, Hercules, Calif.), and equilibrated with 20 ml of 1× loading buffer. The total RNA (1-2 mg) was heated at 65° C. for 8 minutes, quenched on ice for 5 minutes, and after addition of 1 volume of 2× column loading buffer loaded onto the column. The eluate was collected and reloaded 2-3 times by heating the sample as above and quenching on ice prior to each loading. The oligo(dT) column was washed with 10 volumes of 1× loading buffer, then with 3 volumes of medium salt buffer (20 mM Tris-Cl, pH 7.6, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS), followed by elution of the poly(A)⁺ RNA with 3 volumes of elution buffer (10 mM Tris-Cl pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to 65° C., by collecting 500 μl fractions. The absorbance at 260 nm was read for each collected fraction, and the mRNA containing fractions were pooled and ethanol precipitated at −20° C. for 12 hours. The poly(A)⁺ RNA was collected by centrifugation, resuspended in DEPC-treated water, and stored in 5-10 μg aliquots at −80° C.

Double-stranded Eco RI-Not I-directional cDNA was synthesized from 5 μg of Trichoderma reesei RutC30 poly(A)⁺ RNA by the RNase H method (Gubler and Hoffman 1983, Gene 25: 263-270; Sambrook et al., 1989, Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using a hair-pin modification. The poly(A)⁺ RNA (5 μg in 5 μl of DEPC-treated water) was heated at 70° C. for 8 minutes in a pre-siliconized, RNase-free Eppendorf tube, quenched on ice, and combined in a final volume of 50 μl with reverse transcriptase buffer (50 mM Tris-Cl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT) containing 1 mM of dATP, dGTP and dTTP, 0.5 mM of 5-methyl-dCTP, 40 units of human placental ribonuclease inhibitor (Promega, Madison, Wis.), 4.81 μg of oligo(dT)₁₈-Not I primer, and 1000 units of SuperScript II RNase H—reverse transcriptase (Life Technologies, Inc., Rockville, Md.). First-strand cDNA was synthesized by incubating the reaction mixture at 45° C. for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gel filtrated through a MicroSpin S-400 HR spin column (Amersham Biosciences, Piscataway, N.J.) according to the manufacturer's instructions.

After the gel filtration, the hybrids were diluted in 250 μl of second strand buffer (20 mM Iris-Cl pH 7.4, 90 mM KCl, 4.6 mM MgCl₂, 10 mM (NH₄)₂SO₄, 0.16 mM NAD⁺) containing 200 μM of each dNTP, 60 units of E. coli DNA polymerase I (Amersham Biosciences, Piscataway, N.J.), 5.25 units of RNase H, and 15 units of E. coli DNA ligase (New England Biolabs, Inc., Beverly, Mass.). Second strand cDNA synthesis was performed by incubating the reaction tube at 16° C. for 2 hours, and an additional 15 minutes at 25° C. The reaction was stopped by addition of EDTA to 20 mM final concentration followed by phenol and chloroform extractions.

The double-stranded cDNA was ethanol precipitated at −20° C. for 12 hours by addition of 2 volumes of 96% ethanol and 0.2 volume of 10 M ammonium acetate, recovered by centrifugation, washed in 70% ethanol, dried (SpeedVac), and resuspended in 30 μl of Mung bean nuclease buffer (30 mM sodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO₄, 0.35 mM dithiothreitol, 2% glycerol) containing 25 units of Mung bean nuclease. The single-stranded hair-pin DNA was clipped by incubating the reaction at 30° C. for 30 minutes, followed by addition of 70 μl of 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanol precipitation with 2 volumes of 96% ethanol and 0.1 volume 3 M sodium acetate pH 5.2 on ice for 30 minutes.

The double-stranded cDNAs were recovered by centrifugation (30,000×g for 30 minutes), and blunt-ended with T4 DNA polymerase in 30 μl of T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol) containing 0.5 mM of each dNTP, and 5 units of T4 DNA polymerase by incubating the reaction mixture at 16° C. for 1 hour. The reaction was stopped by addition of EDTA to 20 mM final concentration, followed by phenol and chloroform extractions and ethanol precipitation for 12 hours at −20° C. by adding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2.

After the fill-in reaction, cDNA was recovered by centrifugation as above, washed in 70% ethanol, and the DNA pellet was dried in a SpeedVac. The cDNA pellet was resuspended in 25 μl of ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM ATP) containing 2 μg Eco RI adaptors (0.2 μg/μl, Amersham Biosciences, Piscataway, N.J.) and 20 units of T4 ligase (Roche Molecular Biochemicals, Indianapolis, Ind.) by incubating the reaction mix at 16° C. for 12 hours. The reaction was stopped by heating at 65° C. for 20 minutes, and then placed on ice for 5 minutes. The adapted cDNA was digested with Not I by addition of 20 μl autoclaved water, 5 μl of 10× Not I restriction enzyme buffer, and 50 units of Not I, followed by incubation for 3 hours at 37° C. The reaction was stopped by heating the sample at 65° C. for 15 minutes. The cDNAs were size-fractionated by agarose gel electrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarose gel (FMC, Rockland, Me.) in 44 mM Tris Base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer (in autoclaved water) to separate unligated adaptors and small cDNAs. The gel was run for 12 hours at 15 V, and the cDNA was size-selected with a cut-off at 0.7 kb by cutting out the lower part of the agarose gel. Then a 1.5% agarose gel was poured in front of the cDNA-containing gel, and the double-stranded cDNAs were concentrated by running the gel backwards until it appeared as a compressed band on the gel. The cDNA-containing gel piece was cut out from the gel and the cDNA was extracted from the gel using a GFX Gel Band Purification Kit (Amersham, Arlington Heights, Ill.) as follows. The trimmed gel slice was weighed in a 2 ml nuclease-free microcentrifuge tube (ISC BioExpress, Kaysville, Utah) then 10 ml of Capture Buffer (Amersham, Arlington Heights, Ill.) was added for each 10 mg of gel slice, the gel slice was dissolved by incubation at 60° C. for 10 minutes, until the agarose was completely solubilized, and the sample was then pelleted by a brief centrifugation (2 minutes at 8,000×g). The melted sample was transferred to a GFX spin column placed in a collection tube, incubated at 25° C. for 1 minute, and then centrifuged at full speed (15,000×g) in a microcentrifuge for 30 seconds. The flow-through was discarded, and the column was washed with 500 μl of wash buffer (GFX Gel Band Purification Kit, Amersham, Arlington Heights, Ill.) followed by centrifugation at full speed for 30 seconds. The collection tube was discarded, and the column was placed in a 1.5 ml Eppendorf tube, followed by elution of the cDNA by addition of 50 μl of TE pH 7.5 to the center of the column, incubation at 25° C. for 1 minute, and finally by centrifugation for 1 minute at maximum speed (15,000×g). The eluted cDNA was stored at −20° C. until library construction.

A plasmid DNA preparation for a Eco RI-Not I insert-containing pYES2.0 cDNA clone, was purified using a QIAGEN Tip-100 according to the manufacturer's instructions (QIAGEN, Valencia, Calif.). A total of 10 μg of purified plasmid DNA was digested to completion with Not I and Eco RI in a total volume of 60 μl by addition of 6 μl of 10× NEBuffer for Eco RI (New England Biolabs, Beverly, Mass.), 40 units of Not I, and 20 units of Eco RI followed by incubation for 6 hours at 37° C. The reaction was stopped by heating the sample at 65° C. for 20 minutes. The digested plasmid DNA was extracted once with phenol-chloroform, then with chloroform, followed by ethanol precipitation for 12 hours at −20° C. by adding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2. The precipitated DNA was resuspended in 25 μl of TE pH 7.5, loaded onto a 0.8% SeaKem agarose gel in TBE buffer, and run for 3 hours at 60 V. The digested vector was cut out from the gel, and the DNA was extracted from the gel using a GFX Gel Band Purification Kit according to the manufacturer's instructions. After measuring the DNA concentration by absorbance at 260 nm, the eluted vector was stored at −20° C. until library construction.

To establish the optimal ligation conditions for the cDNA library, four test ligations were done in 10 μl of ligation buffer (30 mM Tris-Cl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5 mM ATP) containing 7 μl of double-stranded cDNA (corresponding to approximately 1/10 of the total volume in the cDNA sample), 2 units of T4 ligase, and 25 ng, 50 ng and 75 ng of Eco RI-Not I cleaved pYES2.0 vector, respectively (Invitrogen, Carlsbad, Calif.). The vector background control ligation reaction contained 75 ng of Eco RI-Not I cleaved pYES.0 vector without cDNA. The ligation reactions were performed by incubation at 16° C. for 12 hours, heated at 65° C. for 20 minutes, and then 10 μl of autoclaved water was added to each tube. One μl of the ligation mixtures was electroporated (200 W, 2.5 kV, 25 mF) to 40 μl of electrocompetent E. coli DH10B cells (Life Technologies, Gaithersburg, Md.). After addition of 1 ml of SOC medium (Birren et al, 1998. Genome Analysis, Vol. 2. Detecting Genes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) to each transformation mix, the cells were grown at 37° C. for 1 hour. Then 50 μl and 5 μl from each electroporation were plated on LB plates composed per liter of 10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of NaCl, and 15 g/L of agar, and supplemented with ampicillin at 100 μg per ml and grown at 37° C. for 12 hours. Using the optimal conditions, a Trichoderma reesei RutC30 cDNA library containing 1-2.5×10⁷ independent colony forming units was established in E. coli', with a vector background of ca. 1%. The cDNA library was stored as (1) individual pools (25,000 c.f.u./pool) in 20% glycerol at −80° C.; (2) cell pellets of the same pools at −20° C.; (3) QIAGEN Tip 100 purified plasmid DNA from individual pools at −20° C.; and (4) directional, double-stranded cDNA at −20° C.

Example 3 Trichoderma reesei EST Template Preparation

Plasmid DNAs from individual E. coli colonies from the cDNA libraries described in Example 2 were purified using a 96-well manifold plasmid preparation system (QIAGEN, Valencia, Calif.) according to instructions supplied by the manufacturer.

Example 4 Trichoderma reesei Genomic Library Construction

Genomic DNA from Trichoderma reesei RutC30 cells grown for two days at 34° C. in 100 ml of YEG medium (0.5% yeast extract, 2% glucose) was purified using a DNeasy™ Slant System (QIAGEN, Valencia, Calif.). The DNA was subsequently sheared by nebulization (10 psi, 180 seconds in 2 ml volume), separated by preparative gel electrophoresis (Maniatis et al., 1982. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), and a fraction that was enriched for 2-3 kb fragments was excised and purified with a Gel Extraction Kit (QIAGEN, Valencia, Calif.). The DNA ends were made blunt using Klenow DNA polymerase and dephosphorylated with calf intestinal phosphatase (CIP) (Roche Diagnostics, Indianapolis, Ind.).

The DNA was purified again after CIP treatment and ligated with a pCR4Blunt-TOPO (Invitrogen, Carlsbad, Calif.) vector at molar ratios of 5:1 and 10:1. This cloning reaction was used to transform electrocompetent E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.) and 10 μl of each transformation mix was plated on LB plates containing 100 μg of ampicillin per ml and 250 μg of X-Gal per ml for blue/white screening of recombinants. The transformation efficiencies using 5:1 and 10:1 molar ratios of insert-to-vector were 2.1×10⁶ and 1.8×10⁶ cfu/μg, respectively. Among the transformants, approximately 90% were white and light blue colonies, presumably carrying plasmids with Trichoderma reesei DNA inserts. Ten representative white or light blue colonies from each transformation were used for plasmid DNA preparation and restriction analysis using Eco RI to check the proportion of true recombinants among the transformants. It was found that 7 out of 10 (from the 5:1 molar ratio) and 10 out of 10 (from the 10:1 molar ratio) plasmids carried Trichoderma reesei DNA inserts with sizes ranging from 2-3 kb. Based on the favorable results of the restriction analyses, the remaining E. coli cells were plated from the second ligation mix (insert:vector DNA ratio 10:1). A Qpix robot (Genetix Ltd., Hampshire, UK) was then used to pick the white and light blue colonies into the 96-well plates containing LB medium supplemented with 100 μg of ampicillin per ml. After overnight incubation at 37° C. an equivalent amount of 50% glycerol was added to each well, and the plates were taped with “Petri Seal” and stored at −80° C. for later use. In total, approximately 2×10⁵ colonies were obtained with a colony density of 300-700 per 150 mm plate. Approximately 45,000 colonies were picked, grown overnight in selective medium, and frozen in microtiter plates. This number is sufficient to give about 3× coverage of the genome of Trichoderma reesei (ca. 94% probability of cloning every gene in the genome at least once).

Example 5 Isolation of Trichoderma reesei Genomic Inserts

Individual plasmid DNAs bearing the Trichoderma reesei genomic inserts from each of the clones described in Example 4 were isolated using rolling circle amplification (RCA) using a TempliPhi Kit (Amersham Biosciences, Piscataway, N.J.). The same 96-well plate containing individual clones was used to inoculate a second 96-well plate with one milliliter of LB medium composed per liter of 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl, and supplemented with 100 μg of ampicillin per ml for the conventional plasmid DNA isolation and to perform the RCA. Using RCA, approximately the same amount of high molecular weight DNA product was found in all 96 wells. To assure that this product represented the expected linear multimers of plasmid DNA, all 96 DNA preparations was treated with Eco RI to digest the multimers and to release the insert DNA. In 87 out of 96 digests (91%), both vector and insert DNAs were found. In just 9 samples the Eco RI digestion generated empty vector DNA.

The conditions of rolling circle amplification were optimized for the purpose of DNA microarray production to minimize the volume of starting cell material added to the TempliPhi reaction, as the RCA method is very sensitive to inhibitors from components of saturated culture medium. Moreover, excess of cell material in the reaction results in higher background of cell debris and chromosomal DNA, which might adversely impact signal-to-noise ratios during microarray experiments. Because it is difficult to transfer very small volumes, serial two-fold dilutions (from 2 to 2112) of the original culture material from eight clones in sterile TE buffer were made, and 1 μl from each diluted sample was transferred into a 96-well plate containing Denaturing Buffer (TempliPhi Kit, Amersham Biosciences, Piscataway, N.J.). Following the required incubation period for DNA amplification, one microliter of each RCA product was digested with Eco RI and analyzed by 1% agarose gel electrophoresis using TBE buffer to evaluate the quality of the reactions. The best results (highest yields of DNA and approximately equivalent yields) were obtained with dilutions in the range of 8 to 32 times. Higher dilutions resulted in less consistent DNA yields.

A larger scale trial with 96 clones, using 8-fold dilution, was done as a pilot for future microarray experiments. The RCA products were subsequently digested with Eco RI and analyzed on agarose gels as described above. All samples showed similar yields of DNA product, which was also confirmed by spectrophotometric measurement of DNA concentrations. An average yield of DNA from the RCA reactions was 500 ng/μl. This concentration of DNA allows dilution of the reaction products several times before printing onto microarray slides and avoids a DNA precipitation step, which could result in loss of plasmid DNA.

Example 6 Trichoderma reesei SSH Library Construction

Total cellular RNA was isolated from frozen cells grown on glucose, cellulose, or pre-treated corn stover (Example 1) using slight modifications to the method of Timberlake and Barnard, 1981, Cell 26: 29-37. RNA extraction buffer was prepared by adding a freshly prepared solution of p-aminosalicylic acid (9.6 g in 80 ml of DEPC-treated water) to a solution of triisopropylnaphthalene sulfonic acid (1.6 g in 80 ml of DEPC-treated water). This mixture was added to 40 ml of 5×RNB solution (1 M tris-HCl, pH 8.5, 1.25 M NaCl, 0.25 M EGTA) with stirring. Frozen mycelia were ground to a fine powder in an electric coffee grinder with a few chips of dry ice. The ground mycelia were poured directly into 20 ml of RNA extraction buffer on ice, and an equal volume of TE-saturated phenol was added. After vigorous agitation, the samples were centrifuged at 2500 rpm (Sorvall RT7 centrifuge equipped with a H1000B rotor) for 10 minutes to separate phases. The aqueous phase was transferred to a new tube that contained 10 ml of phenol and 10 ml of chloroform-isoamyl alcohol (24:1), while an additional 5 ml of extraction buffer was added to the phenol phase. The latter mixture was incubated at 68° C. for 5 minutes to liberate RNA trapped in polysomes and in the interface material. Following the incubation, the tubes were centrifuged at 2500 rpm (Sorvall RT7 centrifuge equipped with a H1000B rotor) for 10 minutes and the aqueous phase was combined with that obtained from the first extraction. These mixtures were subjected to repeated extraction with phenol-chloroform until there was no longer protein at the interface (usually five or six times). The RNA was recovered by centrifugation (30 minutes at 12,000×g) following precipitation with 0.3 M sodium acetate pH 5.2 and 50% isopropanol. From each sample consisting of approximately 1-2 grams of frozen mycelia generated in laboratory-scale fermentors, 0.4-1.8 mg of total cellular RNA was obtained.

The quality of RNA from cultures grown on cellulose and PCS was appraised by formaldehyde-agarose gel electrophoresis followed by Northern blotting and hybridization (Thomas, 1980, Proc. Nat. Acad. Sci. USA 77: 5201-5205) with a Trichoderma reesei cbh1 specific probe. The cbh1 probe fragment was amplified by standard PCR methods based on the published nucleotide sequence information available from the EMBL database (accession number E00389). The probes were labeled with horseradish peroxidase (HRP) and hybridized at 55° C. using the buffers and protocols provided in a North2South Direct HRP Labeling and Detection Kit (Pierce, Rockford, Ill.). The blots were washed three times in 2×SSC with 0.1% SDS at 55° C. for five minutes each, followed by three additional washes in 2×SSC (no SDS) for five minutes each. Following exposure of the blot to X-ray film, it was clear that virtually all of the hybridization signal in each lane was contained in a 1.8 kb cbh1 mRNA species that migrated to a position just slightly above the 18S ribosomal RNA band. There was no evidence of significant mRNA degradation on either the autoradiogram or on the ethidium bromide stained gel. Polyadenylated (polyA⁺) mRNA fractions were purified using an Oligotex™ mRNA Isolation Kit according to the manufacturer's instructions (QIAGEN, Valencia, Calif.). Yields of polyA+ mRNA from each of these samples ranged from 2 μg to 25 μg. Each of the mRNA fractions was subsequently analyzed by Northern blot hybridization using HRP-labeled probes derived from the Trichoderma reesei γ-actin and cbh1 genes. The γ-actin probe fragment was amplified by standard PCR methods and the following gene-specific primers.

5′-CCAGACATGACAATGTTGCCGTAG-3′ (SEQ ID NO: 1189) 5′-TTTCGCTCTTCCTCACGCCATTG-3′ (SEQ ID NO: 1190) As expected, the hybridization signals were localized in bands that corresponded to the γ-actin and cbh1 mRNAs (ca. 1.2 kb and 1.8 kb, respectively) in each lane, indicating that the mRNA samples were of high quality and suitable for cDNA synthesis.

The suppression subtractive hybridization (SSH) method described by Diatchenko et al., 1996, supra, was used to generate a cDNA pool from Trichoderma reesei RutC30 that was both enriched for cellulose- and PCS-induced sequences and normalized to aid in recovery of rare transcripts (FIG. 1). Table 1 below lists the combinations of driver and tester cDNAs used for these experiments.

TABLE 1 Driver and tester cDNA pools used for SSH. SSH Reaction Driver cDNA source Tester cDNA source 1 Glucose-grown cells PCS-grown cells 2 Glucose-grown cells Cellulose-grown cells 3 Cellulose-grown cells PCS-grown cells

The resulting cDNA pools from the SSH reactions in Table 1 were used to generate subtractive libraries of cellulose- and PCS-induced sequences. For synthesis of cDNA, 400 ng of polyA⁺ mRNA derived from each time point (1-5 days) was combined for a total of 2 μg of template. Synthesis and subtraction of cDNA was done using a PCR-Select™ Kit (Clontech, Palo Alto, Calif.). The methods are based on the procedure of suppression subtractive hybridization (SSH) as outlined by Diatchenko et al, 1996, supra. The overall scheme is shown in FIG. 1. First, mRNA was converted from three separate fermentations of Trichoderma reesei RutC30 grown on glucose, cellulose, and PCS into double-stranded cDNA using reagents supplied with the PCR-Select™ Kit (Clontech, Palo Alto, Calif.). The differentially expressed cDNAs were present in both the “tester” cDNA pool (i.e., from cells grown on cellulose or corn stover) and the “driver” cDNA, but were present at much lower levels in the “driver” pool (Table 1). Both of these cDNA pools were digested with the restriction enzyme Rsa I which recognizes a four-base pair palindrome and yields blunt-end fragments (GT|AC). The tester cDNA pool was then divided into two samples and ligated with two different adaptor oligonucleotides (provided with the Clontech PCR-Select™ Kit) resulting in two populations of tester cDNA. The adaptors were designed without 5′-phosphate groups such that only the longer strand of each adaptor could be covalently linked to the 5′-ends of the cDNA.

In the first of two hybridizations using conditions specified in the Clontech PCR-Select™ Kit an excess of driver cDNA was added to each portion of tester cDNA. The mixtures were denatured by heating to 95° C. then allowed to anneal. Four types of molecules were generated by this annealing (designated as a, b, c, and d molecules). Type a molecules included equal concentrations of high- and low-abundance cDNAs, because the second-order kinetics of hybridization were faster for more abundant molecules in the pool which preferentially formed b type molecules. At the same time, type a molecules were significantly enriched for differentially expressed (e.g., cellulose- or PCS-induced) sequences, since common non-target cDNAs formed type c molecules with the driver. In a second hybridization, the two pools of primary hybridized products were combined so that the type a molecules from each tester sample could associate and form new type e hybrids. These were double-stranded tester molecules with different adaptor sequences on each end. Fresh denatured driver cDNA was also added to further enrich the pool of e molecules for differentially expressed sequences.

In the final step of the SSH procedure, the differentially expressed cDNAs were selectively amplified by PCR (conditions specified in the PCR-Select™ Kit) Only type e molecules that have two different primer annealing sites were amplified exponentially.

As a quality check, the cDNA clones from approximately 360 randomly picked colonies were purified by rolling circle amplification using an Amersham TempliPhi Kit and analyzed by DNA sequencing (70 from Reaction 1, 96 from Reaction 2, and 192 from Reaction 3). Clustering of the sequences using Transcript Assembler™ software (Paracel, Inc., Pasadena, Calif.) showed that each pool contained a high percentage of non-redundant clones—76% for Reaction 1, 90% for Reaction 2, and 67% for Reaction 3. In addition, the contigs (overlapping sequences of the same cDNA) identified in this analysis contained on average only two sequences. Collectively, these observations suggested that efficient normalization of the libraries was achieved during the SSH reactions, yielding a low level of redundancy in the corresponding cDNA libraries. These differentially expressed sequences were greatly enriched in the final subtracted cDNA pool, and useful as a hybridization probe or to create a subtractive library.

Subtracted and normalized cDNA fractions generated by the SSH procedure were ligated with pCR11-TOPO (Invitrogen, Carlsbad, Calif.) and the ligation mixtures were used to transform electrocompetent E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.). Transformants were selected on LB agar plates (Miller, J. H. 1992. A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) that contained 250 μg/ml X-Gal (no IPTG) and ampicillin at a final concentration of 100 μg/ml.

In order to evaluate the efficiency of subtraction and normalization in SSH cDNA libraries, two approaches were used: colony hybridization and sequencing of random clones from each SSH library. The procedure for colony hybridization is detailed in Birren et at 1998. Genome Analysis, A Laboratory Manual, Vol. 2, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Colony-hybridization analysis included approximately 700 independent clones from each subtracted [PCS minus glucose (SG), cellulose minus glucose (CG), PCS minus cellulose (SC)] and un-subtracted (cellulose, PCS) cDNA libraries with DIG-labeled cbh1 probe (abundant transcript), and γ-actin probe, a moderately abundant transcript representing a house-keeping gene (Table 2).

TABLE 2 Colony hybridization of SSH libraries probed with cbh1 and γ-actin cDNA fragments. Library Frequency cbh1 Frequency γ-actin Cellulose (no SSH) 3.3% <0.17% PCS (no SSH) 3.7% 0.13% PCS minus glucose 0.4% ND Cellulose minus glucose 0.5% ND PCS minus cellulose 0.1% ND ND, not detected.

While the cbh1 was a rather abundant in the non-subtracted cellulose and PCS libraries (3.3% and 3.6% correspondingly), the subtracted SG and CG libraries contained almost 10 times less cbh1 clones, which indicated that the abundant transcript was successfully normalized. Colony-hybridization of the SC library showed very low occurrence of cbh1 (only 0.1% of cbh1 clones) indicating an efficient subtraction of this abundant transcript when performing SSH with cell populations both expressing high levels of cbh1.

The cDNA clones from approximately 360 randomly picked colonies were purified by rolling circle amplification (RCA) (Dean et at, 2001, Genome Res. 11: 1095-1099) and analyzed by DNA sequencing (70 from Reaction 1, 96 from Reaction 2, and 192 from Reaction 3). Clustering of the sequences using Transcript Assembler™ software showed that each pool contained a high percentage of non-redundant clones: 76% for reaction 1, 90% for reaction 2, and 67% for reaction 3. In addition, the contigs (overlapping sequences of the same cDNA) identified in this analysis contained on the average only two sequences. Collectively, these observations suggested that efficient normalization of the libraries was achieved during the SSH reactions, yielding a low level of redundancy in the corresponding cDNA libraries.

Example 7 Identification of Differentially Expressed cDNAs Using Microarrays

Plasmid DNA samples from 3608 colonies representing the three SSH cDNA libraries (1152 clones each from SG and SC libraries, and 1304 clones from the CG library) were prepared using rolling-circle amplification (RCA). RCA (Dean et al, 2001, supra) of plasmid DNA from frozen cells was done using TempiPhi™ reagents (Amersham, Arlington Heights, Ill.). The amplified cDNA clones were diluted to a concentration of 100-400 ng/μl in 3×SSC and spotted from 384-well plates onto poly-L-lysine coated glass microscope slides using equipment and methods that were described previously (Eisen and Brown, 1999, Methods Enzymol. 303: 179-205). Several control DNAs were included as well: cbh1, cbh2, egl1, egl2, serine hydroxymethyl transferase cDNA, γ-actin, and 285 rDNA.

Fluorescent probes were prepared by reverse transcription of poly(A)⁺ RNA, incorporating aminoallyl-dUTP into first strand cDNA (Berka et al., 2003, Proc. Nat. Acad. Sci. USA 100: 5682-5687). The amino-cDNA products were subsequently labeled by direct coupling to either Cy3 or Cy5 monofunctional reactive dyes (Amersham, Arlington Heights, Ill.) and purified as described previously (Berka et al., 2003, supra). In all cases, cDNA from cells grown on glucose was used as the control (Cy3 label), and cDNA from cells grown on cellulose or PCS was labeled with Cy5. Cy3 and Cy5 labeled probes were combined, purified using a QIAquick PCR Purification Kit (QIAGEN, Valencia, Calif.) and dried under vacuum, resuspended in 15.5 μl of water, and combined with the following: 3.6 μl of 20×SSC, 2.5 μl of 250 mM HEPES (pH 7.0), 1.8 μl of poly-dA (500 μg/ml), and 0.54 μl of 10% SDS. Before hybridization, the solution was filtered with a 0.22 μm filter, heated to 95° C. for 2 minutes, and cooled to room temperature.

The fluorescently labeled cDNAs were applied to microarrays under cover glasses, placed in a humidified chamber, and incubated at 63° C. overnight (15-16 hours). Before scanning, the arrays were washed consecutively in 1×SSC with 0.03% SDS, 0.2×SSC, and 0.05×SSC and centrifuged for 2 minutes at 500 rpm to remove excess liquid. Microarray slides were imaged using an Axon GenePix® 4000B Scanner (Axon Instruments, Union City, Calif.), and the fluorescence signals for microarray spots were quantified using GenePix® Pro 5.0 software (Axon Instruments). PMT voltages were adjusted during image collection such that the average ratio of fluorescence intensities for the entire array was approximately 1.0. The S+ ArrayAnalyzer 2.0 microarray analysis software (Insightful Corporation, Seattle, Wash.) was used for analyzing of the microarray data. Raw fluorescence intensity values were normalized using the loess function and differential expression analysis was performed using the LPE test. Those spots that were statistically significant (P<0.1) in the LPE test and for which change in the Cy5:Cy3 intensity ratios were greater than 2.0 were chosen for DNA sequencing analysis.

From the 3608 clones that were screened, 728 cDNAs were found to be differentially expressed in technical replicates with differences in Cy5:Cy3 intensity ratios ≧2.0. This represented a substantial fraction of differentially expressed genes in the SSH libraries (19%) compared to 0.7% found by microarray-based screening of 25,000 random clones from a Trichoderma reesei genomic DNA library (not shown). The distribution of biomass-induced genes among three SSH cDNA libraries is shown in Table 3.

TABLE 3 Biomass-induced cDNAs from subtractive (SSH) libraries Number Cellulose/PCS- Library screened Induced cDNAs PCS minus glucose (SG) 1152 209 Cellulose minus glucose (CG) 1332 429 PCS minus cellulose (SC) 1152 90 TOTAL 3840 728

Example 8 DNA Sequencing and Analysis of Nucleotide Sequence Data of the Trichoderma reesei EST, Genomic, and SSH Libraries

DNA sequencing of the Trichoderma reesei ESTs and SSH clones was conducted with a capillary array sequencer ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Inc., Foster City, Calif.) using ABI-Prism BigDye terminator chemistry (Applied Biosystems, Inc., Foster City, Calif.) and standard M13 forward and reverse primers. Vector sequence and low quality sequence were removed with Phred and crossmatch (University of Washington, Seattle, Wash.). The sequences were assembled with Phrap (University of Washington, Seattle, Wash.). The assembled sequences and singletons were searched with BLAST (Altschul, et al., 1997, Nucleic Acids Res. 25: 3389-3402.) against PIR-NREF, a comprehensive database containing non-redundant protein sequences from PIR-PSD, Swiss-Prot, TrEMBL, RefSeq, GenPept, and PDB.

The sequences of the Trichoderma reesei ESTs and SSH clones are designated SEQ ID NOs. 1-1188. An “N” in a nucleic acid sequence means that the nucleotide is an A, C, G, or T. SEQ ID NOs: 1-24 are the ESTs and SEQ ID NOs: 25-1188 are the SSH clones.

The ESTs and SSH clones were compared by means of computer algorithms for homologies to the content of individual families. All sequences from a given family were used individually as a query to search a database of EST sequences of the invention using a number of different homology search algorithms like FASTA and BLAST (W. R. Pearson, 1990, Rapid and Sensitive Sequence Comparison with FASTP and FASTA, Methods in Enzymology 183: 63-98; and Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David 3. Lipman, 1990, Basic local alignment search tool, Journal of Molecular Biology 215: 403-10). A distinct hit to a sequence of a given family predicted the particular EST or SSH clone sequence to encode a protein of that family. Using this method, part of the EST and SSH clone sequences listed in Table 4 were shown to belong to distinct enzyme families.

TABLE 4 Function Description of ESTs and SSH clones SEQ ID PIR Accession NO. Description Number 1 hypothetical protein FG05562.1 [Gibberella zeae] NF01711769 3 hypothetical protein FG00742.1 [Gibberella zeae] NF01710516 4 hypothetical protein FG01373.1 [Gibberella zeae] NF01713147 5 hypothetical protein FG07555.1 [Gibberella zeae] NF01716785 6 predicted protein [Magnaporthe grisea] NF01573568 7 hypothetical protein FG08771.1 [Gibberella zeae] NF01710529 8 hypothetical protein FG07461.1 [Gibberella zeae] NF01712722 9 hypothetical protein FG01855.1 [Gibberella zeae] NF01708666 12 hypothetical protein FG01513.1 [Gibberella zeae] NF01708403 13 Glucan 1,3 beta-glucosidase-like protein NF00186100 14 hypothetical protein FG06214.1 [Gibberella zeae] NF01708590 15 Cyclophilin-RNA interacting protein (EC 5.2.1.8) NF00948523 20 hypothetical protein FG07072.1 [Gibberella zeae] NF01707147 21 hypothetical protein FG01462.1 [Gibberella zeae] NF01716821 22 hypothetical protein FG09663.1 [Gibberella zeae] NF01712777 23 hypothetical protein FG02234.1 [Gibberella zeae] NF01714687 24 hypothetical protein FG00711.1 [Gibberella zeae] NF01711861 26 Hypothetical protein [Neurospora crassa] NF01483858 29 conserved hypothetical protein [Gibberella zeae] NF01709003 30 conserved hypothetical protein [Gibberella zeae] NF01708228 31 hypothetical protein MG01153.4 [Magnaporthe grisea] NF01578120 32 hypothetical protein FG09234.1 [Gibberella zeae] NF01708819 33 Cip2 [Hypocrea jecorina] NF01379722 35 hypothetical protein FG02467.1 [Gibberella zeae] NF01714575 36 hypothetical protein MG07201.4 [Magnaporthe grisea] NF01578621 37 hypothetical protein FG08610.1 [Gibberella zeae] NF01706364 38 HEX1 [Hypocrea jecorina] NF01538586 39 60S ribosomal protein L12 [Neurospora crassa] NF00649304 42 Exopolygalacturonase PGX1 [Fusarium oxysporum] NF00755681 43 hypothetical protein FG05845.1 [Gibberella zeae] NF01708916 50 hypothetical protein FG10259.1 [Gibberella zeae] NF01713550 51 Hypothetical protein [Neurospora crassa] NF01489009 52 Predicted protein [Neurospora crassa] NF01483451 53 hypothetical protein UM05244.1 [Ustilago maydis] NF01814929 54 hypothetical protein UM05244.1 [Ustilago maydis] NF01814929 55 hypothetical protein UM05244.1 [Ustilago maydis] NF01814929 56 Putative senescence-associated protein (Fragment) NF00726461 [Pisum sativum] 57 Putative senescence-associated protein (Fragment) NF01555598 [Pyrus communis] 58 hypothetical protein UM05244.1 [Ustilago maydis] NF01814929 59 predicted protein [Gibberella zeae] NF01714387 61 Hypothetical protein [Neurospora crassa] NF01486230 63 Probable 26s proteasome p44.5 protein [Neurospora crassa] NF00647520 65 hypothetical protein FG08366.1 [Gibberella zeae] NF01708463 66 hypothetical protein FG09004.1 [Gibberella zeae] NF01714213 70 hypothetical protein FG08493.1 [Gibberella zeae] NF01708515 71 Endoglucanase III [Trichoderma viride] NF01407726 72 Endoglucanase III [Trichoderma viride] NF01407726 73 Endoglucanase III [Trichoderma viride] NF01407726 74 Hypothetical protein [Neurospora crassa] NF01487792 76 hypothetical protein FG05177.1 [Gibberella zeae] NF01712231 78 hypothetical protein FG01986.1 [Gibberella zeae] NF01711586 79 predicted protein [Emericella nidulans] NF01788176 82 Cellobiohydrolase II [Trichoderma viride] NF01470256 83 Cellobiohydrolase II [Trichoderma viride] NF01470256 84 Cellobiohydrolase II [Trichoderma viride] NF01470256 85 Cellobiohydrolase II [Trichoderma viride] NF01470256 86 hypothetical protein FG06119.1 [Gibberella zeae] NF01706228 87 Strain NRRL Y-1140 chromosome D of strain NRRL Y-1140 of NF01870375 Kluyveromyces lactis (Fragment) [Kluyveromyces lactis] 88 Strain NRRL Y-1140 chromosome D of strain NRRL Y-1140 of NF01870375 Kluyveromyces lactis (Fragment) [Kluyveromyces lactis] 94 Similar to tr|Q96WT5 Aspergillus oryzae Maltose permease NF01905886 [Debaryomyces hansenii] 95 Similar to tr|Q96WT5 Aspergillus oryzae Maltose permease NF01905886 [Debaryomyces hansenii] 96 hypothetical protein FG00070.1 [Gibberella zeae] NF01714480 97 Endoglucanase III [Trichoderma viride] NF01407726 98 Endoglucanase III [Trichoderma viride] NF01407726 100 hypothetical protein FG05066.1 [Gibberella zeae] NF01714926 101 conserved hypothetical protein [Gibberella zeae] NF01713888 102 hypothetical protein FG03260.1 [Gibberella zeae] NF01708766 103 hypothetical protein MG04400.4 [Magnaporthe grisea] NF01582694 105 hypothetical protein FG07945.1 [Gibberella zeae] NF01714580 106 Swollenin precursor [Hypocrea jecorina] NF00494342 107 Swollenin precursor [Hypocrea jecorina] NF00494342 108 Swollenin precursor [Hypocrea jecorina] NF00494342 109 Swollenin precursor [Hypocrea jecorina] NF00494342 111 hypothetical protein FG06718.1 [Gibberella zeae] NF01706773 112 COI i1 protein [Agrocybe aegerita] NF00746529 114 hypothetical protein FG06516.1 [Gibberella zeae] NF01713352 117 hypothetical protein FG06239.1 [Gibberella zeae] NF01707463 122 hypothetical protein FG04149.1 [Gibberella zeae] NF01708688 124 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 125 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 134 hypothetical protein MG06524.4 [Magnaporthe grisea] NF01576755 135 hypothetical protein FG10921.1 [Gibberella zeae] NF01708328 137 hypothetical protein AN7772.2 [Emericella nidulans] NF01783212 138 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 139 hypothetical protein AN6831. 2 [Emericella nidulans] NF01781110 140 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 141 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 145 Hypothetical protein [Neurospora crassa] NF01485496 146 hypothetical protein FG03028.1 [Gibberella zeae] NF01714672 147 Cip2 [Hypocrea jecorina] NF01379722 148 hypothetical protein FG03028.1 [Gibberella zeae] NF01714672 150 hypothetical protein FG10320.1 [Gibberella zeae] NF01708223 151 hypothetical protein FG05572.1 [Gibberella zeae] NF01707831 156 hypothetical protein FG03424.1 [Gibberella zeae] NF01713250 157 hypothetical protein FG03424.1 [Gibberella zeae] NF01713250 158 Polyketide synthase [Cochliobolus heterostrophus] NF01679256 160 hypothetical protein FG06645.1 [Gibberella zeae] NF01710877 162 hypothetical protein FG04880.1 [Gibberella zeae] NF01711500 163 Exoglucanase I precursor (EC 3.2.1.91) (Exocellobiohydrolase I) NF00769949 (CBHI) (1,4-beta-cellobiohydrolase) [Hypocrea ceramica] 164 Exoglucanase I precursor (EC 3.2.1.91) (Exocellobiohydrolase I) NF00769949 (CBHI) (1,4-beta-cellobiohydrolase) [Hypocrea ceramica] 166 Tripeptidylpeptidase 2 precursor [Aspergillus fumigatus] NF01739585 168 hypothetical protein FG00916.1 [Gibberella zeae] NF01707379 170 hypothetical protein FG01868.1 [Gibberella zeae] NF01710654 171 Hypothetical protein B24M22.120 [Neurospora crassa] NF00822527 172 CISY_NEUCR Citrate synthase, mitochondrial precursor NF01709377 [Gibberella zeae] 173 Endo-1,4-beta-xylanase 2 precursor (EC 3.2.1.8) (Xylanase 2) NF00494353 (1,4-beta-D-xylan xylanohydrolase 2) [Hypocrea jecorina] 174 Endo-1,4-beta-xylanase (EC 3.2.1.8) IIA [Trichoderma viride] NF00756627 176 Hemolysin [Acanthamoeba polyphaga] NF00003921 178 PCZA361.14 [Amycolatopsis orientalis] NF00428903 179 PCZA361.14 [Amycolatopsis orientalis] NF00428903 180 hypothetical protein AN6396.2 [Emericella nidulans] NF01784171 181 hypothetical protein FG08669.1 [Gibberella zeae] NF01709222 183 Cip1 [Hypocrea jecorina] NF01379724 184 Cellobiohydrolase I [Trichoderma viride] NF01470257 185 ADT_NEUCR ADP, ATP CARRIER PROTEIN (ADP/ATP NF01707425 TRANSLOCASE) (ADENINE NUCLEOTIDE TRANSLOCATOR) (ANT) [Gibberella zeae] 188 Hypothetical protein (Related to aldose 1-epimerase) NF01490813 [Neurospora crassa] 193 40S ribosomal protein S15 (S12) [Podospora anserina] NF00649664 194 hypothetical protein FG05134.1 [Gibberella zeae] NF01714637 195 hypothetical protein FG01081.1 [Gibberella zeae] NF01706654 197 hypothetical protein FG05399.1 [Gibberella zeae] NF01711065 199 hypothetical protein FG01106.1 [Gibberella zeae] NF01705903 200 Related to phenol 2-monooxygenase [Neurospora crassa] NF00649064 204 Abf2 [Hypocrea jecorina] NF01379727 205 Abf2 [Hypocrea jecorina] NF01379727 206 hypothetical protein FG08587.1 [Gibberella zeae] NF01711855 211 hypothetical protein FG09286.1 [Gibberella zeae] NF01714081 212 hypothetical protein FG00248.1 [Gibberella zeae] NF01713168 214 hypothetical protein FG04512.1 [Gibberella zeae] NF01714942 215 Cellobiohydrolase I [Trichoderma viride] NF01470257 216 hypothetical protein AN1677.2 [Emericella nidulans] NF01784923 217 Cellobiohydrolase I [Trichoderma viride] NF01470257 221 Endoglucanase III [Trichoderma viride] NF01407726 222 Endoglucanase III [Trichoderma viride] NF01407726 223 IntI (Fragment) [Citrobacter freundii] NF00904695 224 Acetyl Xylan Esterase [Hypocrea jecorina] NF00494390 225 Acetylxylan esterase precursor (EC 3.1.1.72) [Hypocrea jecorina] NF00494333 226 Acetylxylan esterase precursor (EC 3.1.1.72) [Hypocrea jecorina] NF00494333 227 D-xylose reductase (EC 1.1.1.9) [Hypocrea jecorina] NF01282548 228 conserved hypothetical protein [Gibberella zeae] NF01706258 229 Xylanase III [Hypocrea jecorina] NF00494389 230 HSP70 [Hypocrea jecorina] NF01324889 231 HSP70 [Hypocrea jecorina] NF01324889 233 Neuronal calcium sensor 1 [Magnaporthe grisea] NF00946519 234 QI74 protein [Hypocrea lixii] NF00756616 236 CISY_NEUCR Citrate synthase, mitochondrial precursor NF01709377 [Gibberella zeae] 237 Hypothetical protein [Neurospora crassa] NF01483858 238 Cip2 [Hypocrea jecorina] NF01379722 240 Bete-glucosidase [Hypocrea jecorina] NF00494329 241 Acid phosphatase (Fragment) [Aspergillus niger] NF00626490 242 hypothetical protein FG03882.1 [Gibberella zeae] NF01708914 243 hypothetical protein FG10320.1 [Gibberella zeae] NF01708223 244 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 245 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 246 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 247 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 248 hypothetical protein AN3876.2 [Emericella nidulans] NF01785990 249 Elongation factor 1-alpha (EF-1-alpha) [Hypocrea jecorina] NF00494402 253 hypothetical protein FG02628.1 [Gibberella zeae] NF01706121 254 Hypothetical protein [Neurospora crassa] NF01487111 257 Polyubiquitin [Tuber borchii] NF00176514 259 hypothetical protein FG07555.1 [Gibberella zeae] NF01716785 260 hypothetical protein MG00775.4 [Magnaporthe grisea] NF01577948 261 RRNA intron-encoded homing endonuclease (Fragment) NF00253537 [Oryza sativa] 262 hypothetical protein FG08530.1 [Gibberella zeae] NF01710746 264 Predicted protein [Neurospora crassa] NF01485930 266 hypothetical protein FG07925.1 [Gibberella zeae] NF01706457 269 Repressible high-affinity phosphate permease NF00648186 [Neurospora crassa] 270 Heat shock protein 80 [Neurospora crassa] NF00647533 272 Probable hydroxyacylglutathione hydrolase [Neurospora crassa] NF00648544 273 Hydrophobin II precursor (HFBII) [Hypocrea jecorina] NF00494356 274 Heat shock protein 90 homolog (Suppressor of vegetative NF00649581 incompatibility MOD-E) [Podospora anserina] 275 Probable maturase [Hypocrea jecorina] NF00873684 278 hypothetical protein FG00553.1 [Gibberella zeae] NF01709063 279 hypothetical protein FG09864.1 [Gibberella zeae] NF01713126 283 hypothetical protein FG06097.1 [Gibberella zeae] NF01712468 284 hypothetical protein MG00971.4 [Magnaporthe grisea] NF01574249 285 hypothetical protein FG05259.1 [Gibberella zeae] NF01711539 287 hypothetical protein FG09906.1 [Gibberella zeae] NF01708997 288 Glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8) NF01732006 [Trichoderma atroviride] 290 GR78_NEUCR 78 KDA GLUCOSE-REGULATED PROTEIN NF01712405 HOMOLOG PRECURSOR (GRP 78) (IMMUNOGLOBULIN HEAVY CHAIN BINDING PROTEIN HOMOLOG) (BIP) [Gibberella zeae] 291 hypothetical protein MG06267.4 [Magnaporthe grisea] NF01580275 292 hypothetical protein AN6154.2 [Emericella nidulans] NF01786374 293 hypothetical protein FG07180.1 [Gibberella zeae] NF01713398 294 Similar to sp|P16474 Saccharomyces cerevisiae YJL034w KAR2 NF01905417 nuclear fusion protein [Debaryomyces hansenii] 299 Hypothetical protein [Neurospora crassa] NF01490079 300 hypothetical protein FG02697.1 [Gibberella zeae] NF01712949 301 hypothetical protein FG09164.1 [Gibberella zeae] NF01713596 304 Hypothetical protein [Neurospora crassa] NF01484547 305 hypothetical protein FG04985.1 [Gibberella zeae] NF01706601 306 hypothetical protein FG07382.1 [Gibberella zeae] NF01708643 311 hypothetical protein FG08587.1 [Gibberella zeae] NF01711855 312 hypothetical protein FG08587.1 [Gibberella zeae] NF01711855 313 hypothetical protein AN8186.2 [Emericella nidulans] NF01787561 316 hypothetical protein FG04237.1 [Gibberella zeae] NF01709332 319 hypothetical protein FG06675.1 [Gibberella zeae] NF01706901 321 Serine/threonine kinase IREI [Hypocrea jecorina] NF01379730 323 hypothetical protein AN7772.2 [Emericella nidulans] NF01783212 324 hypothetical protein FG05252.1 [Gibberella zeae] NF01709811 332 hypothetical protein FG06956.1 [Gibberella zeae] NF01715297 333 hypothetical protein FG09403.1 [Gibberella zeae] NF01708030 334 hypothetical protein FG09403.1 [Gibberella zeae] NF01708030 335 Swollenin precursor [Hypocrea jecorina] NF00494342 336 Cel61b [Hypocrea jecorina] NF01379731 338 PCZA361.14 [Amycolatopsis orientalis] NF00428903 339 hypothetical protein FG02725.1 [Gibberella zeae] NF01714298 341 Class V chitinase (Endochitinase class V) [Hypocrea virens] NF00939353 343 conserved hypothetical protein [Gibberella zeae] NF01712388 344 conserved hypothetical protein [Gibberella zeae] NF01716160 345 Hypothetical protein [Neurospora crassa] NF01490310 347 Hypothetical protein ART1 [Saccharomyces cerevisiae] NF00955424 348 hypothetical protein FG00765.1 [Gibberella zeae] NF01712921 350 hypothetical protein AN8789.2 [Emericella nidulans] NF01780447 351 hypothetical protein FG10027.1 [Gibberella zeae] NF01714529 355 hypothetical protein FG05213.1 [Gibberella zeae] NF01708686 357 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 359 hypothetical protein FG10027.1 [Gibberella zeae] NF01714529 360 hypothetical protein FG10027.1 [Gibberella zeae] NF01714529 361 hypothetical protein FG06945.1 [Gibberella zeae] NF01712739 363 Swollenin precursor [Hypocrea jecorina] NF00494342 364 Endo-1,4-B-D-Mannanase [Hypocrea jecorina] NF00494391 367 Hypothetical protein (Related to EDE1 protein) NF01486195 [Neurospora crassa] 369 hypothetical protein FG01191.1 [Gibberella zeae] NF01708050 370 hypothetical protein AN1577.2 [Emericella nidulans] NF01780487 371 hypothetical protein AN1577.2 [Emericella nidulans] NF01780487 373 Cip1 [Hypocrea jecorina] NF01379724 374 hypothetical protein FG11637.1 [Gibberella zeae] NF01712269 376 Cel61b [Hypocrea jecorina] NF01379731 377 hypothetical protein FG05762.1 [Gibberella zeae] NF01709497 380 hypothetical protein FG06113.1 [Gibberella zeae] NF01706743 382 NADH-ubiquinone oxidoreductase chain 1 [Hypocrea jecorina] NF00873695 383 Hypothetical protein (Probable prefoldin subunit 5) NF01487302 [Neurospora crassa] 384 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 385 Acetylxylan esterase precursor (EC 3.1.1.72) [Hypocrea jecorina] NF00494333 386 Acetylxylan esterase precursor (EC 3.1.1.72) [Hypocrea jecorina] NF00494333 387 hypothetical protein FG07476.1 [Gibberella zeae] NF01706432 388 Beta-xylosidase precursor (EC 3.2.1.37) [Hypocrea jecorina] NF00494350 389 hypothetical protein FG03723.1 [Gibberella zeae] NF01713175 391 hypothetical protein FG09287.1 [Gibberella zeae] NF01713279 392 Hypothetical protein [Neurospora crassa] NF01486153 393 hypothetical protein FG10726.1 [Gibberella zeae] NF01706641 394 hypothetical protein FG08743.1 [Gibberella zeae] NF01707411 395 hypothetical protein FG02328.1 [Gibberella zeae] NF01708392 397 Putative serine hydroxymethyltransferase, mitochondrial NF01487447 precursor (EC 2.1.2.1) (Serine methylase) (Glycine hydroxymethyltransferase) (SHMT) [Neurospora crassa] 398 hypothetical protein MG05941.4 [Magnaporthe grisea] NF01580292 400 hypothetical protein FG09129.1 [Gibberella zeae] NF01710763 402 hypothetical protein FG08610.1 [Gibberella zeae] NF01706364 404 hypothetical protein FG05089.1 [Gibberella zeae] NF01713808 406 Related to glucosidase II, alpha subunit [Neurospora crassa] NF01053974 408 Ribosomal protein S5 [Hypocrea jecorina] NF00873692 409 Meiosis-specific topoisomerase Spo11 [Sordaria macrospora] NF01466214 410 Endo-1,4-B-D-Mannanase [Hypocrea jecorina] NF00494391 411 Endoglucanase I [Trichoderma viride] NF01407727 413 Strain NRRL Y-1140 chromosome A of strain NRRL Y-1140 of NF01870073 Kluyveromyces lactis [Kluyveromyces lactis] 414 Cel1b [Hypocrea jecorina] NF01379721 416 hypothetical protein FG06115.1 [Gibberella zeae] NF01706331 417 hypothetical protein FG09775.1 [Gibberella zeae] NF01707684 420 hypothetical protein FG01216.1 [Gibberella zeae] NF01711762 423 hypothetical protein UM05244.1 [Ustilago maydis] NF01814929 424 predicted protein [Emericella nidulans] NF01784780 426 Similarities with sp|Q9Y071 Periplaneta americana Putative NF01867546 transcription factor [Kluyveromyces lactis] 427 predicted protein [Emericella nidulans] NF01784780 428 hypothetical protein MG11015.4 [Magnaporthe grisea] NF01575169 439 Endoglucanase III [Trichoderma viride] NF01407726 440 Cellobiohydrolase II [Trichoderma viride] NF01470256 441 Cellobiohydrolase II [Trichoderma viride] NF01470256 442 Strain NRRL Y-1140 chromosome D of strain NRRL Y-1140 of NF01867244 Kluyveromyces lactis (Fragment) [Kluyveromyces lactis] 443 Strain NRRL Y-1140 chromosome D of strain NRRL Y-1140 of NF01870375 Kluyveromyces lactis (Fragment) [Kluyveromyces lactis] 444 Strain NRRL Y-1140 chromosome D of strain NRRL Y-1140 of NF01870375 Kluyveromyces lactis (Fragment) [Kluyveromyces lactis] 447 hypothetical protein FG08841.1 [Gibberella zeae] NF01706464 450 hypothetical protein MG10519.4 [Magnaporthe grisea] NF01577826 452 Endoglucanase III [Trichoderma viride] NF01407726 453 hypothetical protein FG03260.1 [Gibberella zeae] NF01708766 454 Swollenin precursor [Hypocrea jecorina] NF00494342 455 Swollenin precursor [Hypocrea jecorina] NF00494342 456 Swollenin precursor [Hypocrea jecorina] NF00494342 457 Swollenin precursor [Hypocrea jecorina] NF00494342 463 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 464 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 466 hypothetical protein FG03028.1 [Gibberella zeae] NF01714672 468 hypothetical protein FG03028.1 [Gibberella zeae] NF01714672 470 Endoglucanase III [Trichoderma viride] NF01407726 473 Endoglucanase IV precursor (EC 3.2.1.4) (Endo-1,4-beta- NF00494352 glucanase IV) (Cellulase IV) (EGIV) [Hypocrea jecorina] 474 Endoglucanase IV precursor (EC 3.2.1.4) (Endo-1,4-beta- NF00494352 glucanase IV) (Cellulase IV) (EGIV) [Hypocrea jecorina] 475 conserved hypothetical protein [Gibberella zeae] NF01705663 476 conserved hypothetical protein [Gibberella zeae] NF01705663 478 Exoglucanase I precursor (EC 3.2.1.91) (Exocellobiohydrolase I) NF00769949 (CBHI) (1,4-beta-cellobiohydrolase) [Hypocrea ceramica] 479 Exoglucanase I precursor (EC 3.2.1.91) (Exocellobiohydrolase I) NF00769949 (CBHI) (1,4-beta-cellobiohydrolase) [Hypocrea ceramica] 480 Endoglucanase III [Trichoderma viride] NF01407726 481 Tripeptidylpeptidase 2 precursor [Aspergillus fumigatus] NF01739585 484 hypothetical protein FG09306.1 [Gibberella zeae] NF01716109 485 Hypothetical protein 65E11.040 [Neurospora crassa] NF00647950 486 hypothetical protein FG03590.1 [Gibberella zeae] NF01715080 487 hypothetical protein FG03590.1 [Gibberella zeae] NF01715080 490 40S ribosomal protein S15 (S12) [Podospora anserina] NF00649664 492 hypothetical protein FG01230.1 [Gibberella zeae] NF01706327 494 conserved hypothetical protein [Gibberella zeae] NF01707560 495 Abf2 [Hypocrea jecorina] NF01379727 502 hypothetical protein FG04512.1 [Gibberella zeae] NF01714942 503 Cellobiohydrolase I [Trichoderma viride] NF01470257 504 Cellobiohydrolase II [Trichoderma viride] NF01470256 505 Cellobiohydrolase II [Trichoderma viride] NF01470256 506 Cellobiohydrolase II [Trichoderma viride] NF01470256 507 Endoglucanase III [Trichoderma viride] NF01407726 508 Acetyl Xylan Esterase [Hypocrea jecorina] NF00494390 510 hypothetical protein FG10222.1 [Gibberella zeae] NF01710342 511 hypothetical protein FG10222.1 [Gibberella zeae] NF01710342 517 Bete-glucosidase [Hypocrea jecorina] NF00494329 518 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 519 Hypothetical protein [Neurospora crassa] NF01487111 521 Polyubiquitin [Schizosaccharomyces pombe] NF00300425 522 Polyubiquitin [Gibberella pulicaris] NF00647182 523 Polyubiquitin [Schizosaccharomyces pombe] NF00300425 524 hypothetical protein FG07925.1 [Gibberella zeae] NF01706457 525 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 526 Probable hydroxyacylglutathione hydrolase [Neurospora crassa] NF00648544 530 Similar to sp|P16474 Saccharomyces cerevisiae YJL034w KAR2 NF01905417 nuclear fusion protein [Debaryomyces hansenii] 534 hypothetical protein FG08587.1 [Gibberella zeae] NF01711855 540 hypothetical protein MG03691.4 [Magnaporthe grisea] NF01578801 542 Cel61b [Hypocrea jecorina] NF01379731 546 Hypothetical protein ART1 [Saccharomyces cerevisiae] NF00955424 550 Apocytochrome B [Hypocrea jecorina] NF00873696 551 Intron-encoded DNA endonuclease I-AniI precursor (mRNA NF00176170 maturase bI1) (COB intron protein) [Contains: Truncated, nonfunctional cytochrome b; DNA endonuclease/RNA maturase I-AniI (EC 3.1.—.—)] [Emericella nidulans] 552 cellulose 1,4-beta-cellobiosidase (EC 3.2.1.91) II NF00756633 [Trichoderma viride] 553 Cellobiohydrolase II [Trichoderma viride] NF01470256 554 Hypothetical protein [Neurospora crassa] NF01490857 555 Hypothetical protein [Neurospora crassa] NF01490857 558 Cel61b [Hypocrea jecorina] NF01379731 559 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 560 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 561 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 562 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 563 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 566 conserved hypothetical protein [Gibberella zeae] NF01712388 567 Cytochrome c oxidase polypeptide I [Hypocrea jecorina] NF00873697 569 Putative ABC transporter [Mycosphaerella graminicola] NF00381211 570 Similar to aryl-alcohol oxidase from Pleurotus pulmonarius NF01217365 [Podospora anserina] 573 hypothetical protein FG04382.1 [Gibberella zeae] NF01706345 577 conserved hypothetical protein [Gibberella zeae] NF01716580 578 Hypothetical protein [Neurospora crassa] NF01489642 583 hypothetical protein FG06707.1 [Gibberella zeae] NF01714193 587 conserved hypothetical protein [Gibberella zeae] NF01709891 598 hypothetical protein MG01995.4 [Magnaporthe grisea] NF01575095 601 HEX1 [Hypocrea jecorina] NF01538586 603 hypothetical protein FG05593.1 [Gibberella zeae] NF01706656 605 Hexose transporter [Aspergillus parasiticus] NF00626774 606 hypothetical protein FG06196.1 [Gibberella zeae] NF01715262 607 hypothetical protein FG03126.1 [Gibberella zeae] NF01706334 608 hypothetical protein MG07264.4 [Magnaporthe grisea] NF01573254 617 hypothetical protein FG06108.1 [Gibberella zeae] NF01713127 619 hypothetical protein FG09495.1 [Gibberella zeae] NF01717010 622 hypothetical protein FG09966.1 [Gibberella zeae] NF01714711 636 Similar to sp|P56092 Candida maltosa EPD1 protein NF01906680 [Debaryomyces hansenii] 638 DNA lyase [Cordyceps militaris] NF01250907 639 Endoglucanase I [Trichoderma viride] NF01407727 645 VA0D_NEUCR Vacuolar ATP synthase subunit d (V-ATPase d NF01716692 subunit) (Vacuolar proton pump d subunit) (V-ATPase 41 kDa subunit) [Gibberella zeae] 646 conserved hypothetical protein [Gibberella zeae] NF01707651 647 Stress response element binding protein [Trichoderma atroviride] NF01028245 648 heat shock protein [Aspergillus niger] NF01893378 649 Hypothetical protein (Probable glucokinase) [Neurospora crassa] NF01484367 650 Glucan synthase [Coccidioides posadasii] NF01745740 655 hypothetical protein FG04237.1 [Gibberella zeae] NF01709332 658 hypothetical protein FG03821.1 [Gibberella zeae] NF01712437 667 hypothetical protein MG03494.4 [Magnaporthe grisea] NF01582484 669 hypothetical protein FG10735.1 [Gibberella zeae] NF01712758 672 Enolase (EC 4.2.1.11) (2-phosphoglycerate dehydratase) (2- NF00626668 phospho-D-glycerate hydro-lyase) [Aspergillus oryzae] 680 Hypothetical protein [Neurospora crassa] NF01484177 683 Thiazole biosynthetic enzyme, mitochondrial precursor (Stress- NF00755602 inducible protein sti35) [Fusarium oxysporum] 684 hypothetical protein FG09535.1 [Gibberella zeae] NF01712897 685 Histidine kinase [Magnaporthe grisea] NF00213123 690 conserved hypothetical protein [Gibberella zeae] NF01715974 700 AGL352Wp [Eremothecium gossypii] NF01657112 701 Hypothetical protein [Neurospora crassa] NF01487930 702 hypothetical protein MG00195.4 [Magnaporthe grisea] NF01574164 705 hypothetical protein AN3499.2 [Emericella nidulans] NF01781700 707 Beta-D-glucoside glucohydrolase precursor (EC 3.2.1.21) NF00494358 [Hypocrea jecorina] 709 hypothetical protein FG00700.1 [Gibberella zeae] NF01707186 710 hypothetical protein MG10568.4 [Magnaporthe grisea] NF01579367 715 hypothetical protein FG09381.1 [Gibberella zeae] NF01712438 719 hypothetical protein FG01970.1 [Gibberella zeae] NF01711972 722 hypothetical protein FG10246.1 [Gibberella zeae] NF01708661 723 Hypothetical protein [Neurospora crassa] NF01490566 724 hypothetical protein MG08177.4 [Magnaporthe grisea] NF01576377 726 hypothetical protein FG09566.1 [Gibberella zeae] NF01710505 728 ATP-binding cassette transporter ABC1 [Venturia inaequalis] NF00903329 730 NADH-ubiquinone oxidoreductase chain 1 [Hypocrea jecorina] NF00873695 731 hypothetical protein MG00195.4 [Magnaporthe grisea] NF01574164 736 hypothetical protein AN8551.2 [Emericella nidulans] NF01787890 737 hypothetical protein FG00840.1 [Gibberella zeae] NF01715067 740 hypothetical protein FG02601.1 [Gibberella zeae] NF01711773 741 hypothetical protein FG09721.1 [Gibberella zeae] NF01715057 743 Endoglucanase I [Trichoderma viride] NF01407727 748 hypothetical protein FG00486.1 [Gibberella zeae] NF01713181 761 hypothetical protein MG03220.4 [Magnaporthe grisea] NF01583628 762 hypothetical protein FG02160.1 [Gibberella zeae] NF01712582 763 Glucan synthase [Paracoccidioides brasiliensis] NF00227448 765 EF2_NEUCR Elongation factor 2 (EF-2) (Colonial temperature- NF01706540 sensitive 3) [Gibberella zeae] 766 conserved hypothetical protein [Gibberella zeae] NF01717050 773 hypothetical protein FG03993.1 [Gibberella zeae] NF01706971 774 hypothetical protein FG03994.1 [Gibberella zeae] NF01706918 777 Hypothetical protein (Related to phosphatidyl-ethanolamine N- NF01489841 methyltransferase) [Neurospora crassa] 780 hypothetical protein MG10688.4 [Magnaporthe grisea] NF01578406 781 Hypothetical protein ((AJ296278) putative 60s ribosomal protein) NF01490749 [Neurospora crassa] 785 hypothetical protein FG05901.1 [Gibberella zeae] NF01711268 788 ABC transporter PMR5 [Penicillium digitatum] NF00246136 790 hypothetical protein FG06111.1 [Gibberella zeae] NF01712281 791 hypothetical protein MG06293.4 [Magnaporthe grisea] NF01582543 793 predicted protein [Emericella nidulans] NF01785418 794 hypothetical protein FG07016.1 [Gibberella zeae] NF01708057 796 Hypothetical protein [Neurospora crassa] NF01487960 800 Hypothetical protein [Neurospora crassa] NF01490566 802 Probable inosine triphosphate pyrophosphatase NF01287098 [Neurospora crassa] 805 hypothetical protein MG01504.4 [Magnaporthe grisea] NF01573616 809 hypothetical protein FG08572.1 [Gibberella zeae] NF01714474 810 ADP, ATP carrier protein (ADP/ATP translocase) (Adenine NF00649281 nucleotide translocator) (ANT) [Neurospora crassa] 811 hypothetical protein FG08391.1 [Gibberella zeae] NF01712334 819 Hypothetical protein [Neurospora crassa] NF01485743 820 hypothetical protein MG04719.4 [Magnaporthe grisea] NF01573760 821 hypothetical protein FG05434.1 [Gibberella zeae] NF01716288 823 Probable glutamate synthase (NADPH) [Neurospora crassa] NF00648682 833 Multidrug resistance protein MDR (ABC-transporter) NF00176333 [Emericella nidulans] 835 hypothetical protein FG07453.1 [Gibberella zeae] NF01710295 837 hypothetical protein FG08338.1 [Gibberella zeae] NF01714420 841 hypothetical protein MG03351.4 [Magnaporthe grisea] NF01573271 844 hypothetical protein FG02564.1 [Gibberella zeae] NF01713345 851 hypothetical protein FG07977.1 [Gibberella zeae] NF01706394 853 hypothetical protein FG09253.1 [Gibberella zeae] NF01716450 856 Elongation factor 1-alpha (EF-1-alpha) [Hypocrea jecorina] NF00494402 857 hypothetical protein MG00814.4 [Magnaporthe grisea] NF01577550 860 hypothetical protein FG07778.1 [Gibberella zeae] NF01715768 862 conserved hypothetical protein [Gibberella zeae] NF01713966 867 Probable 40S ribosomal protein S5 [Neurospora crassa] NF01527794 870 Isocitrate lyase (EC 4.1.3.1) (Isocitrase) (Isocitratase) (ICL) NF00649093 [Neurospora crassa] 876 hypothetical protein FG08530.1 [Gibberella zeae] NF01710746 878 Hypothetical protein PFI1335w [Plasmodium falciparum] NF01067476 880 hypothetical protein FG07102.1 [Gibberella zeae] NF01713887 887 hypothetical protein FG09422.1 [Gibberella zeae] NF01714429 893 hypothetical protein MG04989.4 [Magnaporthe grisea] NF01582762 894 hypothetical protein FG03577.1 [Gibberella zeae] NF01707340 896 hypothetical protein FG01219.1 [Gibberella zeae] NF01709359 901 hypothetical protein FG03994.1 [Gibberella zeae] NF01706918 903 hypothetical protein AN1608.2 [Emericella nidulans] NF01784340 909 Hypothetical protein SCO0467 [Streptomyces coelicolor] NF00550337 910 Beta-xylosidase precursor (EC 3.2.1.37) [Hypocrea jecorina] NF00494350 911 hypothetical protein FG09878.1 [Gibberella zeae] NF01713637 913 Probable 40S ribosomal protein S5 [Neurospora crassa] NF01527794 916 NADH-ubiquinone oxidoreductase chain 4 [Hypocrea jecorina] NF00873689 920 Predicted protein [Neurospora crassa] NF01484649 921 hypothetical protein FG06754.1 [Gibberella zeae] NF01710230 925 hypothetical protein FG03072.1 [Gibberella zeae] NF01709591 927 Clock-controlled protein 6 [Neurospora crassa] NF00647506 933 Hypothetical protein [Neurospora crassa] NF01485547 937 hypothetical protein FG10782.1 [Gibberella zeae] NF01707462 940 Probable maturase [Hypocrea jecorina] NF00873684 941 Hypothetical protein [Neurospora crassa] NF01488438 944 hypothetical protein MG10181.4 [Magnaporthe grisea] NF01577807 946 Hypothetical protein H4H7.110 [Neurospora crassa] NF01485963 949 hypothetical protein FG06588.1 [Gibberella zeae] NF01710623 952 Hypothetical protein (Probable glucokinase) [Neurospora crassa] NF01484367 953 hypothetical protein FG01154.1 [Gibberella zeae] NF01707852 955 conserved hypothetical protein [Gibberella zeae] NF01714033 956 Hypothetical protein [Neurospora crassa] NF01489636 958 QI74 protein [Hypocrea lixii] NF00756616 961 hypothetical protein AN3199.2 [Emericella nidulans] NF01781622 970 Endo-1,4-beta-xylanase (EC 3.2.1.8) IIB (Proteinase-sensitive) NF00756643 [Trichoderma viride] 972 hypothetical protein FG07946.1 [Gibberella zeae] NF01716616 975 60S ribosomal protein L16 [Neurospora crassa] NF00648465 977 Frequency clock protein [Hypocrea spinulosa] NF00426736 978 hypothetical protein FG00578.1 [Gibberella zeae] NF01715708 981 Hypothetical protein [Neurospora crassa] NF01486656 982 hypothetical protein FG05145.1 [Gibberella zeae] NF01707421 984 hypothetical protein FG06394.1 [Gibberella zeae] NF01716035 988 hypothetical protein FG06328.1 [Gibberella zeae] NF01706390 989 hypothetical protein FG04412.1 [Gibberella zeae] NF01717062 991 hypothetical protein FG09233.1 [Gibberella zeae] NF01709847 993 hypothetical protein AN2875.2 [Emericella nidulans] NF01787595 994 hypothetical protein FG03563.1 [Gibberella zeae] NF01705877 995 hypothetical protein FG07011.1 [Gibberella zeae] NF01715546 996 Apocytochrome B [Hypocrea jecorina] NF00873696 1002 hypothetical protein FG04512.1 [Gibberella zeae] NF01714942 1004 hypothetical protein FG05467.1 [Gibberella zeae] NF01714488 1015 NADH dehydrogenase subunit 3 (NADH-ubiquinone NF00494385 oxidoreductase chain 3) [Hypocrea jecorina] 1020 Hypothetical protein [Neurospora crassa] NF01483424 1022 Hypothetical protein [Neurospora crassa] NF01485488 1023 hypothetical protein FG10920.1 [Gibberella zeae] NF01711642 1027 hypothetical protein FG07245.1 [Gibberella zeae] NF01708545 1028 hypothetical protein FG01586.1 [Gibberella zeae] NF01709463 1029 Hypothetical protein [Neurospora crassa] NF01485668 1030 conserved hypothetical protein [Gibberella zeae] NF01715089 1033 hypothetical protein FG00854.1 [Gibberella zeae] NF01714634 1034 hypothetical protein FG08125.1 [Gibberella zeae] NF01706145 1036 hypothetical protein FG10345.1 [Gibberella zeae] NF01714154 1037 hypothetical protein FG01861.1 [Gibberella zeae] NF01716331 1038 hypothetical protein FG01843.1 [Gibberella zeae] NF01711037 1039 hypothetical protein FG06003.1 [Gibberella zeae] NF01708535 1041 Plasma membrane H+-ATPase [Aspergillus fumigatus] NF00795376 1043 hypothetical protein AN6831.2 [Emericella nidulans] NF01781110 1044 hypothetical protein MG05487.4 [Magnaporthe grisea] NF01578435 1049 hypothetical protein FG05066.1 [Gibberella zeae] NF01714926 1052 hypothetical protein MG05197.4 [Magnaporthe grisea] NF01582047 1054 hypothetical protein FG06356.1 [Gibberella zeae] NF01715584 1056 hypothetical protein FG01606.1 [Gibberella zeae] NF01714118 1060 Probable HDEL receptor ERD2 [Neurospora crassa] NF00903991 1063 hypothetical protein MG04438.4 [Magnaporthe grisea] NF01577745 1065 RAN_BRUMA GTP-binding nuclear protein RAN/TC4 [Gibberella zeae] NF01708935 1067 hypothetical protein FG10303.1 [Gibberella zeae] NF01707774 1068 hypothetical protein FG01656.1 [Gibberella zeae] NF01708271 1069 hypothetical protein FG07120.1 [Gibberella zeae] NF01713195 1075 hypothetical protein FG04392.1 [Gibberella zeae] NF01710243 1077 Cytochrome P450 oxidoreductase [Gibberella fujikuroi] NF01399523 1079 hypothetical protein FG01297.1 [Gibberella zeae] NF01715472 1080 hypothetical protein FG08463.1 [Gibberella zeae] NF01710966 1081 hypothetical protein FG00644.1 [Gibberella zeae] NF01706083 1082 hypothetical protein FG08610.1 [Gibberella zeae] NF01706364 1084 hypothetical protein FG09229.1 [Gibberella zeae] NF01711344 1086 hypothetical protein MG03911.4 [Magnaporthe grisea] NF01579335 1089 Cytochrome c oxidase polypeptide II [Hypocrea jecorina] NF00873681 1090 Endoglucanase IV precursor (EC 3.2.1.4) (Endo-1,4-beta- NF00494352 glucanase IV) (Cellulase IV) (EGIV) [Hypocrea jecorina] 1098 Similar to tr|Q96WT5 Aspergillus oryzae Maltose permease NF01905886 [Debaryomyces hansenii] 1101 hypothetical protein FG04027.1 [Gibberella zeae] NF01714863 1104 hypothetical protein FG05231.1 [Gibberella zeae] NF01713188 1106 Beta-xylosidase precursor (EC 3.2.1.37) [Hypocrea jecorina] NF00494350 1107 hypothetical protein FG08312.1 [Gibberella zeae] NF01714073 1111 hypothetical protein FG02406.1 [Gibberella zeae] NF01711411 1119 Beta-tubulin 2 [Hypocrea virens] NF01248224 1120 ATP synthase protein 9, mitochondrial precursor (EC 3.6.3.14) NF00648607 (Lipid- binding protein) [Neurospora crassa] 1124 hypothetical protein FG04387.1 [Gibberella zeae] NF01716461 1125 Probable adenylate kinase (EC 2.7.4.3) (ATP-AMP NF00648385 transphosphorylase) [Neurospora crassa] 1126 hypothetical protein FG10717.1 [Gibberella zeae] NF01705725 1127 Hypothetical protein [Neurospora crassa] NF01490566 1128 PMA1_NEUCR Plasma membrane ATPase (Proton pump) NF01716280 [Gibberella zeae] 1129 hypothetical protein FG05853.1 [Gibberella zeae] NF01707732 1131 hypothetical protein FG07218.1 [Gibberella zeae] NF01709286 1133 hypothetical protein FG09492.1 [Gibberella zeae] NF01715612 1136 hypothetical protein FG06073.1 [Gibberella zeae] NF01712503 1138 hypothetical protein FG01544.1 [Gibberella zeae] NF01715994 1141 Hypothetical protein B2G14.120 [Neurospora crassa] NF01287101 1142 hypothetical protein FG09052.1 [Gibberella zeae] NF01707043 1143 Class V chitin synthase [Glomerella graminicola] NF01209406 1144 hypothetical protein FG01262.1 [Gibberella zeae] NF01706669 1146 Alpha-glucuronidase precursor (EC 3.2.1.139) (Alpha- NF00494377 glucosiduronase) (GLRI) [Hypocrea jecorina] 1147 Hypothetical protein [Neurospora crassa] NF01487149 1148 Hypothetical protein (Related to brefeldin A resistance protein) NF01485464 [Neurospora crassa] 1153 conserved hypothetical protein [Gibberella zeae] NF01712603 1155 Probable maturase [Hypocrea jecorina] NF00873685 1158 Endo-1,4-beta-xylanase 1 precursor (EC 3.2.1.8) (Xylanase 1) NF00494326 (1,4-beta-D-xylan xylanohydrolase 1) [Hypocrea jecorina] 1160 hypothetical protein FG06931.1 [Gibberella zeae] NF01710995 1165 hypothetical protein FG06756.1 [Gibberella zeae] NF01707441 1168 Putative endo-exoxylanase [Leptosphaeria maculans] NF01286326 1168 Putative endo-exoxylanase [Leptosphaeria maculans] NF01286326 1170 Acetyl Xylan Esterase [Hypocrea jecorina] NF00494390 1171 hypothetical protein FG04827.1 [Gibberella zeae] NF01705701 1172 hypothetical protein MG00373.4 [Magnaporthe grisea] NF01579634 1173 Hypothetical protein [Neurospora crassa] NF01486658 1174 Acetyl Xylan Esterase [Hypocrea jecorina] NF00494390 1176 alpha-glucoside permease [Saccharomyces cerevisiae] NF00363631 1177 hypothetical protein FG10292.1 [Gibberella zeae] NF01717020 1181 hypothetical protein MG01710.4 [Magnaporthe grisea] NF01582967 1188 Hypothetical protein (Related to berberine bridge enzyme) NF00649183 [Neurospora crassa]

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. 

1. A substrate comprising an array of Trichoderma reesei ESTs or SSH clones, or a combination thereof, selected from the group consisting of SEQ ID NOs. 1-1188 for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells. 